Information

Translation of mRNA?


We inserted an insulin gene from human into bacteria. Will translation of the gene (protein formation) occur in bacteria? If translation occurs then why does it occurs, give a reason for this?


Yes, bacteria will produce human (or any organism's) proteins if you introduce their genetic material but there are a few things to consider.

First, the introns must be removed from the human genetic sequence. Bacteria do not have the machinery to splice out introns after transcription. This is typically done by using a viral protein to reverse transcribe mRNA, which already have the introns removed, into DNA which you can use to transform bacteria.

Second, you need to insert a Shine-Dalgarno sequence. This allows the bacterial ribosomes to bind to the mRNA they will produce, and translate the protein you desire. This sequence is not found in eukaryotic genes, so you have to make sure it was included either in the vector or in the PCR primers you used to amplify the gene sequence.


Although the genetic code is almost conserved among organisms there are some issues to take in account, among which:

  • Every organism has its own codon usage, i.e. the differences in frequency of occurrence in synonymous codons in coding DNA (that reflect the composition of tRNA abundances). So, for example, the codons UUU and UUC both encode for phenylalanine but - I'm making this up - UUU is common in human but rare in coli while UUC the contrary. If the coding sequence of insuline is rich of UUU, you probably need to change the codon UUU to UUC in order to avoid slow translation.
  • Also the aminoacid abundance can be very variable between two organisms. One of the ways you can handle the differences is by "tuning" the expression of the aminoacids. Here a table of aminoacids abundance in coli.
  • The regulatory sequences in coli are not the same as in human. In fact, prokaryotic translation initiation needs a purine-rich sequence upstream the (usually) AUG initiation codon called Shine Dalgarno. This sequence is complementary to the 16S RNA in 30S ribosome subunit.
  • Often peptide chains are subjected to various post-translational modifications. The insulin peptide, for example, once synthetized is cleaved by specific peptidases and disulfide bonds are formed. Because human and coli post-translational modification mechanisms are not the same, this can be a difficult problem to handle. Instead of trying to exactly reproduce the protein with all its modifications, often people search for functionally (and not structurally) identical proteins.
  • Also the folding process can be different, both assisted (by chaperones) and spontaneous (withouth chaperones). The folding can be influenced by a number of factors like molecular crowding, pH,…
  • A lot of eukaryotic genes have introns: sequences removed before translation. Once splicing sites are known, this problem is easily solved by using a sequence withouth introns.

All the organisms share a common ancestor and have evolved from it. So they share quite the same machinery for transcription and translation. Codons are also interpreted in the same way in almost all the organisms.

So, yes translation should happen ( Assuming that the gene has been cloned in the right way.)


It works, and is actually done to make all the "human" insulin which is used by diabetes patients today. This is because the genetic code is almost completely conserved (with only very few exceptions in nature), so bacteria use the same codons to code for an amino acid as in humans. So with a suitable vector (a ring of DNA which carries extrachromosomal genetic information) which has the ability to replicate independent from the cell and which has the correct promoters (start points) for initiating transcription in bacterial cells, you can basically clone and express any human gene.


Like any polymerization in a cell, translation occurs in three steps: initiation brings a ribosome, mRNA and an initiator tRNA together to form an initiation complex. Elongation is the successive addition of amino acids to a growing polypeptide. Termination is signaled by sequences (one of the stop codons) in the mRNA and protein termination factors that interrupt elongation and release a finished polypeptide. The events of translation occur at specific A, P and E sites on the ribosome (see drawing below).


Transfer RNAs

During translation, each of the 20 amino acids must be aligned with their corresponding codons on the mRNA template. All cells contain a variety of tRNAs that serve as adaptors for this process. As might be expected, given their common function in protein synthesis, different tRNAs share similar overall structures. However, they also possess unique identifying sequences that allow the correct amino acid to be attached and aligned with the appropriate codon in mRNA.

Transfer RNAs are approximately 70 to 80 nucleotides long and have characteristic cloverleaf structures that result from complementary base pairing between different regions of the molecule (Figure 7.1). X-ray crystallography studies have further shown that all tRNAs fold into similar compact L shapes, which are likely required for the tRNAs to fit onto ribosomes during the translation process. The adaptor function of the tRNAs involves two separated regions of the molecule. All tRNAs have the sequence CCA at their 3´ terminus, and amino acids are covalently attached to the ribose of the terminal adenosine. The mRNA template is then recognized by the anticodon loop, located at the other end of the folded tRNA, which binds to the appropriate codon by complementary base pairing.

Figure 7.1

Structure of tRNAs. The structure of yeast phenylalanyl tRNA is illustrated in open 𠇌loverleaf” form (A) to show complementary base pairing. Modified bases are indicated as mG, methylguanosine mC, methylcytosine DHU, dihydrouridine (more. )

The incorporation of the correctly encoded amino acids into proteins depends on the attachment of each amino acid to an appropriate tRNA, as well as on the specificity of codon-anticodon base pairing. The attachment of amino acids to specific tRNAs is mediated by a group of enzymes called aminoacyl tRNA synthetases, which were discovered by Paul Zamecnik and Mahlon Hoagland in 1957. Each of these enzymes recognizes a single amino acid, as well as the correct tRNA (or tRNAs) to which that amino acid should be attached. The reaction proceeds in two steps (Figure 7.2). First, the amino acid is activated by reaction with ATP to form an aminoacyl AMP synthetase intermediate. The activated amino acid is then joined to the 3´ terminus of the tRNA. The aminoacyl tRNA synthetases must be highly selective enzymes that recognize both individual amino acids and specific base sequences that identify the correct acceptor tRNAs. In some cases, the high fidelity of amino acid recognition results in part from a proofreading function by which incorrect aminoacyl AMPs are hydrolyzed rather than being joined to tRNA during the second step of the reaction. Recognition of the correct tRNA by the aminoacyl tRNA synthetase is also highly selective the synthetase recognizes specific nucleotide sequences (in most cases including the anticodon) that uniquely identify each species of tRNA.

Figure 7.2

Attachment of amino acids to tRNAs. In the first reaction step, the amino acid is joined to AMP, forming an aminoacyl AMP intermediate. In the second step, the amino acid is transferred to the 3´ CCA terminus of the acceptor tRNA and AMP is released. (more. )

After being attached to tRNA, an amino acid is aligned on the mRNA template by complementary base pairing between the mRNA codon and the anticodon of the tRNA. Codon-anticodon base pairing is somewhat less stringent than the standard A-U and G-C base pairing discussed in preceding chapters. The significance of this unusual base pairing in codon-anticodon recognition relates to the redundancy of the genetic code. Of the 64 possible codons, three are stop codons that signal the termination of translation the other 61 encode amino acids (see Table 3.1). Thus, most of the amino acids are specified by more than one codon. In part, this redundancy results from the attachment of many amino acids to more than one species of tRNA. E. coli, for example, contain about 40 different tRNAs that serve as acceptors for the 20 different amino acids. In addition, some tRNAs are able to recognize more than one codon in mRNA, as a result of nonstandard base pairing (called wobble) between the tRNA anticodon and the third position of some complementary codons (Figure 7.3). Relaxed base pairing at this position results partly from the formation of G-U base pairs and partly from the modification of guanosine to inosine in the anticodons of several tRNAs during processing (see Figure 6.38). Inosine can base-pair with either C, U, or A in the third position, so its inclusion in the anticodon allows a single tRNA to recognize three different codons in mRNA templates.

Figure 7.3

Nonstandard codon-anticodon base pairing. Base pairing at the third codon position is relaxed, allowing G to pair with U, and inosine (I) in the anticodon to pair with U, C, or A. Two examples of abnormal base pairing, allowing phenylalanyl (Phe) tRNA (more. )


Affiliations

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, NY, USA

Sulagna Das & Robert H. Singer

Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, New York, NY, USA

Sulagna Das & Robert H. Singer

Department of Biochemistry, McGill University, Montreal, Quebec, Canada

Janelia Research Campus of the HHMI, Ashburn, VA, USA

Valentina Gandin & Robert H. Singer

Systems Biology Lab, Amsterdam Institute of Molecular and Life Sciences (AIMMS), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands

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Contributions

S.D., V.G., E.T. and M.V. researched data for the Review S.D., R.H.S., E.T. and M.V. made a substantial contribution to discussion of content all of the authors wrote the article and S.D., R.H.S. and E.T. reviewed and edited the manuscript before submission.

Corresponding authors


Quality Control

Defective mRNA molecules can be produced by mutations in the gene as well as errors introduced during transcription (albeit at a remarkably low rate). In addition to producing mRNAs with incorrect codons for amino acids, these errors can produce mRNA molecules that have

  • Premature Termination Codons (PTCs) that is, the introduction of a STOP codon before the normal end of the message. Translation of these mRNAs produces a truncated protein that is probably ineffective and may be harmful. The problem can sometimes be solved by Nonsense-Mediated mRNA Decay (NMD).
  • no STOP codon. These produce "nonstop" transcripts. The problem can be solved by Nonstop mRNA Decay.

Nonsense-Mediated mRNA Decay (NMD)

Premature termination codons (PTCs) may be generated by "nonsense" mutations, frameshifts, and RNA processing (intron removal) errors. They are also an inevitable consequence of creating antigen receptors on B cells and T cells.

Mechanisms

  • During RNA processing within the nucleus, protein complexes are added at each spot where adjacent exons are spliced together. (These are important signals for exporting the mRNA to the cytoplasm.)
  • In the cytoplasm, as the ribosome moves down the mRNA, these complexes are removed (and sent back to the nucleus for reuse).
  • If the ribosome encounters a premature termination codon, the final exon-exon tag(s) are not removed, and this marks the defective mRNA for destruction (in P bodies).

Mutations that introduce premature termination codons are responsible for some cases of such inherited human diseases as cystic fibrosis and Duchenne muscular dystrophy (DMD).

A drug, designated PTC124 or ataluren, causes the ribosome to skip over PTCs while still enabling normal termination of translation. PTC124 has shown promise in animal models of cystic fibrosis and DMD and phase II clinical trials are now being conducted on humans.

Nonstop mRNA Decay

Nonstop transcripts occur when there is no STOP codon in the message. As a result the ribosome is unable to recruit the release factors needed to leave the mRNA. Nonstop transcripts are formed during RNA processing, e.g., by having the poly(A) tail put on before the STOP codon is reached.

Mechanisms

Eukaryotes and bacteria handle the problem of no STOP codon differently.

  • In eukaryotes, when the ribosome stalls at the end of the poly(A) tail, proteins are recruited to release the ribosome for reuse and to degrade the faulty message.
  • In bacteria, a special RNA molecule &mdash called tmRNA saves the day. It is called tmRNA because it has the properties of both a transfer RNA and a messenger RNA. The transfer part adds alanine to the A site on the ribosome. The ribosome then moves on to the messenger part which encodes 10 amino acids that target the molecule for destruction (and releases the ribosome for reuse).

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon . The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code .

Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations therefore, a given amino acid is encoded by more than one nucleotide triplet ([Figure 2]).

Figure 2: This figure shows the genetic code for translating each nucleotide triplet, or codon, in mRNA into an amino acid or a termination signal in a nascent protein. (credit: modification of work by NIH)

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons . Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5′ end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.


Part 2: P-bodies and the mRNA Cycle

00:00:00.00 Hello. My name is Roy Parker. I am Professor at the University of Arizona,
00:00:04.08 and an investigator at the Howard Hughes Medical Institute.
00:00:06.26 In my two talks today, I'll be talking about the life of the eukaryotic mRNA.
00:00:10.29 In my first talk, I discussed the mechanisms by which mRNAs are translated,
00:00:15.11 localized, and degraded in eukaryotic cells. In this talk, I'll be discussing P-bodies,
00:00:22.01 and what we call the mRNA cycle.
00:00:25.01 And what that tells us about the regulation of eukaryotic mRNAs.
00:00:31.23 This slide here shows a human liver cell in culture,
00:00:37.26 and you can see these bright regions in the cytoplasm, and these red dots.
00:00:42.03 And these are what we call P-bodies, or cytoplasmic processing bodies.
00:00:46.23 And what we have learned from studying these processing bodies,
00:00:50.11 is that they tell us about a dynamic cycle of mRNAs in the cytoplasm,
00:00:54.15 which affects their function.
00:00:55.27 And that cycle is cartooned in this slide, and has the following key properties.
00:01:01.14 First, mRNAs can exist in a translating state, where they are associated with ribosomes,
00:01:06.05 making proteins.
00:01:07.05 But under certain conditions they can exit that state,
00:01:09.29 lose their translation factors and ribosomes,
00:01:13.04 and assemble what we call a P-body mRNP,
00:01:15.20 which is a translationally repressed mRNP,
00:01:17.29 Which can aggregate in these larger P-bodies.
00:01:20.28 mRNAs within P-bodies can either be destroyed,
00:01:25.00 or, they can return to translation by assembly of a new translation initiation complex,
00:01:30.04 and then recruitment of a ribosome.
00:01:33.02 Now, there are three features of this cycle in P-bodies
00:01:37.11 that I find particularly interesting, and worthy of study.
00:01:41.10 First, the transitions between these different states
00:01:44.19 in the cell are highly likely to play important roles in the regulation of translation
00:01:49.17 and degradation, and perhaps even localization of RNAs in the cell.
00:01:53.16 Obviously, if mRNAs are associated with ribosomes and translation factors
00:01:57.15 they can make proteins. But when they are assembled in translation repression complexes
00:02:01.23 and associated with RNA-degradative complexes,
00:02:05.02 they are more likely to be targeted for destruction.
00:02:07.10 Moreover, we also think that this might play some role in localization,
00:02:11.09 because these components in these P-body structures,
00:02:14.06 actually show similarity to RNA-transport granules in a variety of different cases.
00:02:20.24 So this cycle, probably plays a significant role in just the regulation
00:02:24.21 of translation, degradation, and localization of RNAs in cells.
00:02:29.04 A second interesting feature of this cycle,
00:02:33.01 is that P-bodies, a key component of this cycle,
00:02:37.07 show overlap with other RNA-protein granules that are very important in biology.
00:02:42.08 For example, maternal RNA-granules, or germinal granules,
00:02:47.00 are complexes of maternal mRNAs and proteins found in the oocytes or the embryos,
00:02:53.12 of a wide variety of species. And those maternal mRNAs
00:02:56.11 are made by the mother and then during the development of the embryo,
00:02:59.28 are translated in specific places and at specific times,
00:03:03.21 in order to traject the development of that early embryo.
00:03:07.04 And so the storage, and subsequent translation,
00:03:09.18 at the right time and place is extremely important.
00:03:11.16 And those granules share many components with P-bodies,
00:03:16.03 which are found in every somatic cell that has been examined so far,
00:03:19.07 from yeast to humans.
00:03:22.00 Similarly, in neuronal granules,
00:03:23.19 which play an important role in synaptic plasticity,
00:03:25.29 and transport RNAs out to various synapses, also share components
00:03:32.02 with P-bodies. And also share components with germinal granules.
00:03:35.20 So all three of these types of RNA-granules are related,
00:03:38.21 and probably have an underlying, similar, biochemical and compositional function.
00:03:46.17 Finally, the third reason that I find these interesting,
00:03:49.07 are connections between viruses and the mRNA cycle.
00:03:52.01 So in several cases, components of P-bodies,
00:03:55.19 or stress granules, another granule in this cycle we will talk about,
00:03:58.21 are required for viral life cycles.
00:04:00.21 For example, components of P-bodies are required for
00:04:04.29 retrotransposons, which are retroviral-like elements in yeast.
00:04:09.25 And components of stress granules,
00:04:11.20 such as the protein called DDX-3, is required for translation of HIV
00:04:16.13 genomic RNAs, as well as for hepatitis C viral life cycles.
00:04:22.00 And we think these genetic roles are actually important,
00:04:26.08 because in some cases the viral complexes, or host anti-viral factors
00:04:32.19 accumulate in these structures. So for example, what we are looking at here,
00:04:36.11 these are yeast cells, and the green are actually markers of P-bodies,
00:04:40.25 and the red here, are actually showing newly assembled viral particles
00:04:46.17 for this retrotransposon Ty3. And you can see, while they don't overlap completely,
00:04:52.08 these viral particles tend to be found in conjunction with partial overlap with P-bodies.
00:04:59.20 So my lab has then been interested in trying to understand
00:05:02.20 what the properties and functions of P-bodies are,
00:05:05.29 and how it relates to these dynamic transitions that RNAs can undergo,
00:05:09.16 in regulating their translation and degradation.
00:05:15.15 Now, work from a wide variety of labs has identified many of the components
00:05:21.05 in P-bodies, although we still do not understand the entire complex
00:05:24.14 that is present in these particles.
00:05:26.28 So from yeast to mammals, there is a core set of proteins,
00:05:30.27 which include decapping enzymes,
00:05:33.03 as I talked about in my other talk, plays a role in degrading RNAs
00:05:37.10 by decapping. As well as various proteins which either can repress translation,
00:05:41.08 or promote decapping, as well as an exonuclease,
00:05:44.14 which degrades the RNA following decapping.
00:05:46.26 In some metazoan cells, microRNA components can also be present in P-bodies,
00:05:52.17 or in a related structure, referred to as a GW-body,
00:05:56.19 which is similar to P-bodies and can show some overlap.
00:06:01.03
00:06:03.21 Now P-bodies are proportional to the pool of untranslated RNAs,
00:06:07.09 and I just want to illustrate this, showing the dynamics of these structures.
00:06:11.15 So this is looking at yeast cells during mid-log growth.
00:06:15.02 And you can see there is a few P-bodies of moderate size,
00:06:17.10 in those cells. However, if we starve those cells for glucose,
00:06:21.13 this leads to a rapid loss of translation, and you can see that the P-bodies
00:06:25.18 get quite a bit larger. Conversely, if we treat cells with cyclohexamide,
00:06:31.09 which traps mRNAs in polysomes,
00:06:33.16 that it is in association with ribosomes, the P-bodies disappear.
00:06:37.15 And these type of observations are some of the data which has lead to the model
00:06:43.07 that RNAs partition between translation, or P-bodies, depending upon on whether
00:06:48.08 they are associated with ribosomes or with these components of P-body structures.
00:06:55.00 P-bodies also contain RNA, and we can detect that either by in situ hybridization,
00:07:00.07 or by a common technique which is using the ability to tether GFP to a specific RNA,
00:07:06.14 through sequence specific RNA-binding proteins.
00:07:09.00 Essentially making a GFP-tagged RNA molecule.
00:07:11.13 And if you do that, and express that in cells which are happily growing,
00:07:16.13 here, the RNA tends to be distributed around the cytoplasm,
00:07:19.28 because it is engaged in translation.
00:07:22.06 And we can see that because this is what is called a polysome trace,
00:07:25.07 where the larger of these series of peaks here, these are RNA associated with ribosomes.
00:07:29.26 The larger this peak is here, the more translation that is going on in the cell.
00:07:33.10 Now if we starve that cell for a few minutes with glucose,
00:07:36.06 you can see that translation declines dramatically, these polysomes are now all gone.
00:07:40.22 And now you can see these mRNAs accumulate
00:07:43.15 in these discrete foci within the cell. And those foci overlap with markers of P-bodies.
00:07:48.09 So P-bodies contain RNAs, and those RNAs can actually
00:07:51.21 reversibly leave P-bodies and re-enter translation.
00:07:55.29 Through a number of different experiments
00:07:57.17 shown by Muriel McGees and Daniella Texiera in my lab several years ago.
00:08:02.25 And you can see that if you add glucose back,
00:08:04.17 these P-bodies shrink back down and the polysomes are restored.
00:08:09.01
00:08:11.15 P-bodies don't only contain RNA, but they also require RNA for formation.
00:08:16.02 Here is an experiment done by Marco Valencia Antonio Sanchez in my lab a few years ago,
00:08:22.01 where he purified P-bodies by differential centrifugation,
00:08:25.01 and then if he treated those P-bodies with RNase, you can see that they fell apart.
00:08:29.17 And so P-bodies not only. so P-bodies are complexes that form on non-translating RNA,
00:08:36.11 that contain this discrete set of proteins, and they require that RNA for their formation.
00:08:43.22 So one of the questions my lab has been interested in, is trying to understand,
00:08:46.21 how do these proteins actually assemble onto the RNA,
00:08:50.11 and how do they assemble into a higher order P-body.
00:08:52.06 And what does that tell us about the function of these complexes.
00:08:54.26 And so one approach we have been taking to this,
00:08:57.09 is to actually purify all these different components,
00:08:59.15 and then test for their interactions between them.
00:09:02.10 And we have done a number of different experiments where we purify
00:09:06.00 common versions of these core components, and then test their binding in vitro.
00:09:10.20 For example here, we're taking two different proteins and mix them,
00:09:14.07 we purify this Dhh1 protein back out,
00:09:17.28 and we ask if this one comes along.
00:09:19.16 And if you do a lot of this kind of co-immunoprecipitation experiments,
00:09:22.14 which you can see, is there is a tremendous number of interactions between
00:09:27.08 these core P-body components, and with each other.
00:09:30.07 And with the RNA molecule.
00:09:33.13 So, this cartoon is showing all these components, using purified proteins,
00:09:39.09 showing the direct protein-protein interactions that occur,
00:09:42.15 between these different core components of P-bodies.
00:09:45.13 And what you can see is, there is a dense network of interactions.
00:09:48.02 And within that, many of these proteins are also RNA binding proteins,
00:09:52.09 and so that these proteins can not only assemble with each other,
00:09:54.25 they can also then bind to the RNA molecule to make an RNA-protein complex.
00:09:59.10
00:10:03.00 Now, we don't really know how those come together to form the definitive complex yet.
00:10:09.00 That's one of our goals in the next few years.
00:10:12.05 But, we currently have two models for what these structures look like.
00:10:16.03 One is what we called the closed loop.
00:10:19.00 where the 5-prime and 3-prime end of the RNA are brought together
00:10:21.21 by protein-protein interactions with these different core components,
00:10:25.07 being preferentially bound to the cap, or the 3-prime end of the mRNA.
00:10:29.00 And this kind of closed-loop model is analogous to models for translation complexes,
00:10:34.07 where the cap and poly-A tail are brought together by protein-protein interactions,
00:10:38.15 initiation factors bound to those two sites, to promote the loading of ribosomes.
00:10:45.02 The other possible model is what we call a nucleosome-like,
00:10:48.19 or beads on a string, where we have this core complex, and it's found in multiple places on the RNA,
00:10:54.16 and so the coding region will be coated as well with these different proteins,
00:10:58.27 and current experiments in my lab our directed at trying to identify the differences, to determine
00:11:04.17 which of these complexes actually forms on the RNA.
00:11:09.05
00:11:12.08 Now we understand how these RNAs come together into higher-order structures,
00:11:15.05 from our analysis of these interactions between these proteins.
00:11:19.00 So there are these P-body mRNPs that come together to form a larger structure,
00:11:24.28 through two self interaction domains.
00:11:26.23 So one of these interaction domains is on the EDC3 protein,
00:11:30.18 which can actually dimerize with itself, and so if you disrupt that interaction,
00:11:36.00 what you can see is that you lose. the number of P-bodies goes down dramatically.
00:11:40.16 Although you can still form a few.
00:11:42.08 So this would be a wild-type cell, where we have starved it to make really big P-bodies,
00:11:46.04 and now when we remove that EDC3 protein, you can see there are still some formed.
00:11:51.11 But the ones that formed are dependent on another interaction, which is in the LSM-4 protein,
00:11:56.13 which at its C-terminal tail contains what's called a "prion" domain.
00:12:00.10 OK. And a prion domain is analogous to what we think about in Mad Cow disease,
00:12:05.19 or human Kuru disease, that is a self assembly domain,
00:12:09.12 which at least in the disease state, can be irreversible.
00:12:13.09 But, this is an example where these types of prion domains not only form an aggregate,
00:12:21.02 but that is actually reversible assembly, so it's not a pathogenic state.
00:12:24.20 And if you get both of the EDC3 protein and the prion domain on the LSM4 protein,
00:12:30.15 down here, you see that there are no P-bodies forming anymore.
00:12:34.03 Now, is that interesting that there is actually this prion domain,
00:12:39.25 or what is often called a Q/N domain involved in aggregation of these RNA-protein complexes?
00:12:44.26 I just want to make a few points about this,
00:12:47.19 because I think it is actually a very interesting aspect of the biology,
00:12:50.20 which might have broader significance.
00:12:52.16 First, many proteins involved in translation and RNA-degradation
00:12:57.22 have these type of Q/N domains.
00:12:59.21 For example, if you search the yeast genome,
00:13:02.26 there are about 100 proteins that have the potential
00:13:05.23 to form these type of aggregates through the Q/N or prion domain.
00:13:10.10 Of those 100, 50 are involved in translation or RNA-degradation.
00:13:15.20 So over half. And many of the others we don't know what they do,
00:13:18.11 so they might also be involved.
00:13:20.03 So I think this is going to be a common feature of many of these proteins,
00:13:23.21 in RNA translation and degradation
00:13:26.16 and suggests that they may then have a tendency to form these type of aggregates,
00:13:31.04 for biological reasons we don't understand yet.
00:13:33.09
00:13:35.24 Consistent with that, different types of these domains
00:13:39.13 may create different types of structures, or different types of P-bodies, for example.
00:13:45.17 And, it is not well understood how specific these different Q/N domains can be,
00:13:52.09 but in some cases it appears they can have specific interactions with each other,
00:13:57.08 and in other cases general. So what that means is different types of Q/N domains
00:14:00.28 then could also drive different types of domains, or types of RNA-protein granules.
00:14:05.17 And consistent with that, we also know that Q/N domains
00:14:08.07 are involved in the assembly of another
00:14:10.11 RNA-protein granule in yeast and in human cells called a stress granule.
00:14:14.19 Which I'll discuss in a few minutes.
00:14:16.11
00:14:19.00 Finally, the fact that Q/N domains can be irreversible,
00:14:22.23 allows them to function as epigenetic elements. And what that means then,
00:14:26.23 is that if you assemble RNA-protein granules through these prion domains,
00:14:32.00 you have the potential then to have an heritable genetic state, because of that.
00:14:37.18 And so, a model from Kazeksai and Eric Kandel has proposed that such type of prion driven assembly
00:14:46.05 may play a role in synaptic plasticity in memory formation.
00:14:49.20
00:14:52.17 And then finally, it is striking that several of the neuro-degenerative diseases
00:14:57.16 involve the expansion of poly-glutamine
00:15:01.14 tracts in proteins which are normally components of P-bodies or stress granules.
00:15:05.04 So for example, Huntington's:
00:15:08.01 the protein which gives rise to the neuro-degenerative disease has a polyQ tract in it
00:15:16.14 and it is normally a component of P-bodies. And that is probably why it has the polyQ tract,
00:15:21.06 because part of its biology requires it to assemble into that complex,
00:15:25.04 but when that tract expands too big, it leads to the formation of cytoplasmic aggregates.
00:15:31.09 And it's controversial as to whether those are toxic or protective,
00:15:34.21 but there is a striking correlation between some of these neuro-degenerative diseases,
00:15:38.18 having expansions of these tracts, and the formation of these aggregates,
00:15:43.08 which are probably related in some manner to P-bodies and/or stress granules.
00:15:50.00
00:15:54.16 Alright. So, we understand somewhat then how these P-bodies assemble.
00:16:00.03 And we understand how they aggregate into larger structures.
00:16:03.10 So, this allows us to do an experiment, then to test what is the significance
00:16:08.01 of making these larger structures. Is this larger structure important
00:16:12.24 for these RNAs to stop translation, or is it important for them to be degraded?
00:16:16.24 So, the experiment we have done then, is to simply look at what happens, to RNA-degradation
00:16:24.19 in a mutant that can no longer assemble these larger structures.
00:16:27.25
00:16:29.21 And what we observed then,
00:16:31.16 if we look at degradation of RNAs, is that RNAs tend to degrade pretty normally.
00:16:36.13 So here we are looking at specific reporter mRNA MFA2.
00:16:40.11 We block transcription at 0 time,
00:16:42.06 and you can see that in wild-type cells the RNA degrades with a half life of about 3 minutes.
00:16:46.22 And in the mutant, which can't make large P-bodies, we see a similar decay rate.
00:16:52.08
00:16:55.09 So in other words, formation of these larger scale structures is not required,
00:16:59.17 at least for the degradation of a few reporter mRNAs.
00:17:03.01 And so, that suggests that the formation of these individual mRNPs,
00:17:07.02 at least under the conditions that we have examined so far,
00:17:10.10 is sufficient for allowing RNAs to be degraded, for allowing them to exit translation,
00:17:16.03 and enter this translationally repressed state. So this raises the question then,
00:17:21.27 why do cells make these larger structures?
00:17:25.12 In fact, one has to anticipate that these structures have roles,
00:17:29.24 because they are conserved throughout eukaryotic cells that have been looked at.
00:17:33.16 So, why make these larger structures?
00:17:36.04 The simplest explanation is that you make these larger structures to promote interactions,
00:17:43.15 between components when those components are limiting.
00:17:45.20 And it is very likely under the conditions that we have examined so far in the lab,
00:17:49.29 the components that we are testing, under the conditions that we are examining in the lab,
00:17:56.00 the key components that trigger RNA-degradation, or translation repression, are not limiting.
00:18:02.09 Alternatively, it could be that some mRNAs require aggregation for their regulation.
00:18:07.28 It could be that you aggregate to protect against, to limit other interactions.
00:18:13.13 For example, aggregation of RNA into these structures might protect them from other nucleases,
00:18:18.03 which are not present in those. And then, it could be that aggregation plays important roles
00:18:24.08 in cellular organization, and or transport. As we have suggested in neurons or in germ cells.
00:18:32.10 But this is actually an ongoing area of interest, trying to understand the larger role of assembly,
00:18:40.09 of these large structures in eukaryotic cells. And I want to point out,
00:18:44.20 that this is not a problem that is limited to the study of P-bodies and
00:18:48.14 the regulation of translation in the cytoplasm.
00:18:50.21 In fact, as we have studied more about the organization of cells,
00:18:54.24 what we have learned is that there is a diversity of RNA-protein granules,
00:18:58.08 that form in eukaryotic cells.
00:19:01.04 So, in the cytoplasm we have talked about P-bodies, and a little bit about stress granules.
00:19:05.18 But in the nucleus, there are a lots of other RNA-protein complexes.
00:19:09.15 Nuclear speckles which contain splicing factors, and also Cajal bodies,
00:19:15.02 which contain components of snRNA, small nuclear RNA protein complexes.
00:19:21.04 And actually the work of Carl Neuberger has really shown that the function of these Cajal bodies,
00:19:25.17 is to increase the rate of the assembly of these protein RNA-complexes.
00:19:30.06 And so I think, that one think we should expect as we go forward,
00:19:33.17 as we study these different RNA-protein complexes,
00:19:37.23 is that their general role might be to increase the assembly rates
00:19:42.00 of components within them, simply by providing higher level concentration of those factors.
00:19:49.26
00:19:50.24 Now, P-bodies often dock, or overlap, with these RNA-granules referred to as stress granules.
00:19:59.06 So here, we are looking at a mammalian cell.
00:20:01.15 The blue represents a stress granule structure.
00:20:04.18 And the yellow or red here, represents a P-body.
00:20:08.19 And you can see they are often docked together, near each other.
00:20:11.08 A similar phenomenon occurs in yeast,
00:20:13.12 although in that case, and in many cases, the P-body and stress granule actually overlap.
00:20:19.04 So here, in this particular image in yeast, P-bodies would be red
00:20:23.11 and stress granules would be green.
00:20:26.04 So if they are overlapped, the will be yellow.
00:20:27.16 And you can see that many of these components are in fact yellow.
00:20:30.10
00:20:32.08 So, what are stress granules?
00:20:34.18 So stress granules, again, are an RNA-protein complex containing untranslating RNAs.
00:20:39.21 But in contrast to the decay, and translation repressors present in P-bodies,
00:20:44.24 stress granules contain translation initiation factors,
00:20:47.22 RNA-binding proteins, and, in some cases, they can contain the 40s subunit.
00:20:54.17 Stress granules form when translation initiation is slow,
00:20:58.23 and so the simplest model is that these stress granules represent a population of mRNAs,
00:21:05.16 which are entering or exiting translation.
00:21:07.24 And that is they have assembled a translation initiation complex,
00:21:11.04 but they haven't entered the elongation phase of translation.
00:21:16.16 It's not known whether they are really entering or exiting translation,
00:21:20.02 although in some cases I'm going to argue that they are perhaps entering translation.
00:21:25.01
00:21:27.20 Now, these interactions between P-bodies and stress granules,
00:21:31.06 have lead to the suggestion that mRNAs exchange between these two.
00:21:34.29 And so the logic here is that, while these two different granules interact,
00:21:39.03 they can contain the same mRNA,
00:21:42.28 and we know that mRNAs which are in P-bodies can return to translation.
00:21:46.24 So at some level, those mRNAs must be able to re-associate with translation initiation factors.
00:21:51.20 And so one hypothesis then,
00:21:54.04 is that mRNAs are exchanging between stress granules, and P-bodies.
00:21:57.28
00:22:00.00 And there is really two models for how this could be occurring.
00:22:03.12 So in the first model, mRNAs, when they are engaged in translation,
00:22:10.15 when they cease translation they assemble into this stress granule structure.
00:22:14.08 And within this stress granule, different mRNAs would assemble different complexes.
00:22:19.06 Some RNAs would be targeted for degradation and would be sent to a P-body,
00:22:22.29 for destruction.
00:22:24.14 And other RNAs would reassemble a new translation complex and return to polysomes.
00:22:28.20 The other possibility is, in fact, RNAs when they exit translation they travel to a P-body,
00:22:36.18 and within that P-body some RNAs would be sorted for destruction.
00:22:40.19 Or other RNAs, presumably due to their composition or sequence features,
00:22:45.05 would reassemble a new translation complex,
00:22:47.26 and then would return back to the translated pool.
00:22:50.15 A few years ago, Ross Buck in my lab wanted to try to distinguish between these two models,
00:22:56.28 and so it's relatively a simple experiment,
00:22:59.19 cause he is going to analyze how defects in the ability to assemble P-bodies or stress granules
00:23:04.08 affect the other structures.
00:23:06.16 So for example in this model, you would have to assemble a stress granule
00:23:11.13 in order to make a P-body. Where as in this model, you might have to make a P-body
00:23:15.22 in order to make a stress granule.
00:23:17.02 And so, here's what those kinds of experiments look like.
00:23:21.09 Here we are using yeast cells,
00:23:23.00 where we have a mutation which can prevent the formation of either stress granules or P-bodies.
00:23:27.16 And this is a wild type cell, and again you can see lots of stress granules in green,
00:23:32.13 many P-bodies in red. And they are generally overlapping the stress granule markers,
00:23:37.05 so they are yellow.
00:23:38.16 If you get rid of the ability to form stress granules,
00:23:42.04 you still make P-bodies, and you make about the same number,
00:23:46.02 and the same brightness and the same size of P-bodies.
00:23:51.03 So, mutations reducing stress granules do not affect the formation of P-bodies,
00:23:55.27 at least in yeast cells.
00:23:57.16 However, if we do the converse experiment, where we get rid of P-bodies,
00:24:02.26 and here we are using those mutations we talked about earlier,
00:24:05.10 the EDC3 protein gone and deleting the prion domain on this Lsm4 protein.
00:24:10.16 Now what you see, is that we don't make any P-bodies,
00:24:14.00 but we also don't make any stress granules.
00:24:15.17
00:24:17.04 So our interpretation of that, is in fact, that stress granules form,
00:24:22.07 from pre-existing P-bodies. Now of course, there is two possible views of this:
00:24:28.14 one view is, in fact, that this effect is really an indirect effect.
00:24:32.25 But if you get rid of P-bodies, you have all kinds of changes in the regulation of mRNAs,
00:24:37.13 and that changes the proteins which affect stress granule formation.
00:24:41.03 And therefore you can't make stress granules for some indirect reason.
00:24:44.09 And the other model, is that there in fact, that P-bodies provide an assembly site,
00:24:49.12 where in this model, when RNAs stop translating,
00:24:53.27 they first assemble into these complexes that aggregate in P-bodies, and that with time,
00:24:59.24 some of these RNAs are targeted for translation,
00:25:03.08 so they assemble new translation factors on them,
00:25:06.18 and some are targeted for degradation, and they might go away,
00:25:08.26 and so in this intermediate time we kind of have an overlap,
00:25:12.07 and then with more time, these RNAs are being destroyed, are gone,
00:25:16.26 and these RNAs, which are going to re-enter translation,
00:25:20.06 will become a greater component of the total pool.
00:25:23.23 So in order to look at this, distinguish these possibilities,
00:25:28.18 Ross did an experiment where he basically looked to see if stress granules form
00:25:36.20 by maturation of P-bodies. And so what he is going to do is just follow a time course
00:25:40.29 of the formation of P-bodies and stress granules during a glucose deprivation.
00:25:45.26 So he is going to grow a culture, he's going to starve it for glucose for a short period of time,
00:25:50.13 and then take images over a variety of time,
00:25:53.24 and see what happens to P-bodies and stress granules.
00:25:56.01 And so when he does that experiment, these are the results:
00:26:00.12 The important observations, I'm just going to highlight.
00:26:03.26 The first is, that the first thing you see is that P-bodies get very big.
00:26:08.01 So, from zero time here is before the stress,
00:26:11.27 there are some small P-bodies you can't see under these kind of exposure conditions.
00:26:15.09 But by seven minutes of stress they are quite large,
00:26:18.01 and very bright.
00:26:20.05 So P-bodies form first.
00:26:21.29 The second thing you see, is that the first stress granules you can see,
00:26:26.04 there is a few of them here at this seven-minute time-point,
00:26:29.13 they are always in association with a P-body.
00:26:32.14 So if you look down here in the merge, they always overlap.
00:26:35.04 So stress granules first appear in conjunction with a P-body.
00:26:39.21 And then the third thing you see, is if you follow with more time,
00:26:43.27 some of these P-bodies mature from being a P-body to being primarily like a stress granule.
00:26:50.08 So if you look at this one here, at the early time points, it's primarily a P-body
00:26:54.15 with very little stress granule marker. And then by half an hour later,
00:26:58.18 the amount of Dcp2, a P-body marker there has declined dramatically,
00:27:03.15 and the amount of poly-A binding protein, a stress granule marker has increased,
00:27:07.27 suggesting that these, in fact, are maturing from a P-body state into a stress granule state.
00:27:13.02 And that suggests that there is really a directionality in the movement of RNAs,
00:27:18.29 between these different types of complexes. That they go from translation,
00:27:23.14 to a repressed state, generally in the formation of a P-body, type of complex.
00:27:30.24 And those RNAs can then re-enter translation by forming a new translation complex,
00:27:35.09 which can associate into these larger structures referred to as stress granules,
00:27:39.07 and go back into translation. Although I'm focusing on these individual RNA-protein complexes,
00:27:47.11 we can observe that in the microscope really is a summation of that total population
00:27:52.00 is in these aggregates that are visible in the light microscope.
00:27:56.13
00:28:00.23 Now, so what then are stress granules?
00:28:03.14 So at least under these kind of conditions,
00:28:06.06 this suggest that stress granules are primarily RNAs,
00:28:09.12 which are being primed for re-entering translation.
00:28:12.00 And that then, maybe stress granules should be thought of as sites of assembly,
00:28:18.07 of translation initiation complexes.
00:28:20.06 And the argument here for this is that they form when initiation is limiting,
00:28:24.04 they form from non-translating RNAs, and they contain translation factors.
00:28:28.25 And so maybe they are not necessarily sites where RNAs are targeted for repression,
00:28:32.16 but they are sites where RNAs have their high local concentration of translation factors,
00:28:38.00 and therefore allow for the efficient assembly of these translation initiation complexes
00:28:42.05 which can then go on and enter translation.
00:28:44.17 And this is an area of further research in my lab, as well as other labs, as well.
00:28:50.05
00:28:55.09 Now, I want to step back and just say a few words about RNA granules.
00:28:59.22 Because those of you who work in this area, or read about it,
00:29:04.19 it's easy to become confused about the diversity of different types of RNA granules,
00:29:09.06 which are described in the literature.
00:29:10.24 And, what I'd like to argue is in fact, that maybe these granules are all related,
00:29:16.03 in a simple kind of model where there are different stages
00:29:19.04 of movements of RNAs through this mRNA cycle.
00:29:24.08 Now, so for example, under some conditions you can see granules that accumulate,
00:29:32.25 which contain EIF4e, EIF4g, the cap-binding complex, and poly-A binding protein,
00:29:37.25 that are missing other components,
00:29:40.12 like 40s subunits which form under a different stress.
00:29:44.13 So, a simple way of thinking about these, is that they are simply blocked,
00:29:49.12 they simply have different rate limiting steps in the transitions
00:29:52.13 between these different pools in the mRNA cycle.
00:29:55.09 So if you block this step here,
00:29:57.04 in response to a various cue, these RNAs accumulate with these complexes,
00:30:01.25 then you will get a stress granule of that composition. Whereas if you block over here,
00:30:05.28 you'll accumulate with this type of RNA-protein complex,
00:30:09.02 and you'll get a stress granule which contains these other factors.
00:30:12.06 So, these aren't fundamentally different particles perhaps,
00:30:17.07 they are simply different stall points on a continuum of exchange points.
00:30:22.21 And so when one thinks about that kind of model then,
00:30:26.19 the diversity and the composition of granules observed in the cell
00:30:30.23 can also be affected by the behavior of different mRNA sub-populations.
00:30:36.06 So for example, under some stresses you get both P-body increasing,
00:30:42.20 and stress granules. But probably because there are some mRNAs which are stalled at this stage,
00:30:48.21 and they are assembled with these P-body components, you see a large formation of P-bodies,
00:30:54.01 but other mRNAs are stalled at a different site.
00:30:56.09 Perhaps over here after they have a small subunit,
00:31:00.11 and so you will also see stress granules accumulate.
00:31:02.22 And so one of the things we don't understand very well,
00:31:05.04 is what is the diversity of different types of mRNAs, and mRNA protein complexes,
00:31:09.12 and then how they lead to the accumulation of different particles under different conditions.
00:31:17.15
00:31:19.16 But one thing consistent with this, is if we look in the literature,
00:31:23.15 what we can see is that there is really a continuum of RNA-protein granules,
00:31:27.07 which span from a classical P-body as defined by the initial papers, to a classical stress granule.
00:31:34.19 And so this is just a cartoon of that, and the important thing to look at here,
00:31:38.26 is underneath each of these particles, is shown a list of the proteins found in them,
00:31:44.09 and in green would be components which are only seen in P-bodies.
00:31:47.28 So this would be a P-body which has only components of P-bodies.
00:31:51.13 Yellow are proteins which can be seen in either P-bodies or stress granules.
00:31:55.21 And red would be components which are only seen in stress granules.
00:31:59.11 And if you look around the literature, dendritic P-bodies,
00:32:03.16 these are RNA-protein complexes found
00:32:06.10 at the dendritic side of synapses in various neurons.
00:32:10.08 They have a mixture of both P-bodies and stress granule components.
00:32:14.10 Transport particles in neurons in Drosophila have a mixture again.
00:32:21.10 In C. elegans, different types of granules can again have a mixture.
00:32:25.11 Although in many cases we do not know the complete composition of each of these granules,
00:32:29.12 what you begin to see is that there is no such thing as, there are two boundary conditions,
00:32:35.02 but then there is a wide range of different intermediate forms.
00:32:39.14 And that we should perhaps think of these then,
00:32:42.25 all as different stall points on this mRNA cycle,
00:32:45.22 or different sub-populations of mRNAs stalled at a mixture of sites.
00:32:49.25
00:32:51.24 Alright. So in summary then, I just want to try to highlight the main points that I've tried to say
00:32:58.16 which is that, in cells, mRNAs can exist in at least two predominate mRNP states
00:33:04.12 those that are translating, and those that are repressed.
00:33:07.13 Those repressed mRNAs typically accumulate in RNA-protein granules,
00:33:11.11 such as P-bodies and stress granules.
00:33:13.21 These different biochemical compartments, can have different rates of translation,
00:33:20.08 deadenylation, and mRNA degradation, depending upon where the mRNA is in the cell,
00:33:25.22 and what proteins it is associated with.
00:33:27.07 P-bodies may also be related to RNA transport particles,
00:33:31.09 and therefore play some role in localization,
00:33:33.02 and that there are mechanisms which move RNAs between these compartments,
00:33:36.24 which I haven't had time to talk about today,
00:33:39.03 which are discreet and involve various types of proteins and enzymes.
00:33:43.15
00:33:45.20 There are lots of unanswered questions in this area.
00:33:48.15 How do mRNAs actually cease translation and assemble into P-bodies?
00:33:52.19 How do mRNAs get out of P-bodies and reassemble a new translation complex?
00:33:56.25 And get back into the translating pool?
00:33:59.05 How do they determine which mRNAs are going to do which?
00:34:01.23 And why do you actually form these large scale aggregates?
00:34:05.03 The prediction is, to promote interactions between limiting components,
00:34:08.21 but there is no direct demonstration of that for P-bodies or stress granules yet.
00:34:14.16 And then, how does this cycle relate to the localization of mRNAs to certain regions of the cell
00:34:19.15 and the biogenesis of mRNAs in the nucleus,
00:34:21.26 and their entry into the cytoplasm and beginning to translate and function?
00:34:26.18 And with that, I'd like to thank you all for your attention.
00:34:30.29

  • Part 1: mRNA Localization, Translation and Degradation

Translation

The synthesis of proteins is one of a cell’s most energy-consuming metabolic processes. In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform a wide variety of the functions of a cell. The process of translation, or protein synthesis, involves decoding an mRNA message into a polypeptide product. Amino acids are covalently strung together in lengths ranging from approximately 50 amino acids to more than 1,000.

The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. The composition of each component may vary across species for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNA) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors ([link]).

In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is bonded to its correct amino acid.

The Genetic Code

To summarize what we know to this point, the cellular process of transcription generates messenger RNA (mRNA), a mobile molecular copy of one or more genes with an alphabet of A, C, G, and uracil (U). Translation of the mRNA template converts nucleotide-based genetic information into a protein product. Protein sequences consist of 20 commonly occurring amino acids therefore, it can be said that the protein alphabet consists of 20 letters. Each amino acid is defined by a three-nucleotide sequence called the triplet codon. The relationship between a nucleotide codon and its corresponding amino acid is called the genetic code.

Given the different numbers of “letters” in the mRNA and protein “alphabets,” combinations of nucleotides corresponded to single amino acids. Using a three-nucleotide code means that there are a total of 64 (4 × 4 × 4) possible combinations therefore, a given amino acid is encoded by more than one nucleotide triplet ([link]).

Three of the 64 codons terminate protein synthesis and release the polypeptide from the translation machinery. These triplets are called stop codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also serves as the start codon to initiate translation. The reading frame for translation is set by the AUG start codon near the 5' end of the mRNA. The genetic code is universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all life on Earth shares a common origin.

The Mechanism of Protein Synthesis

Just as with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small ribosome subunit, the mRNA template, three initiation factors, and a special initiator tRNA. The initiator tRNA interacts with the AUG start codon, and links to a special form of the amino acid methionine that is typically removed from the polypeptide after translation is complete.

In prokaryotes and eukaryotes, the basics of polypeptide elongation are the same, so we will review elongation from the perspective of E. coli. The large ribosomal subunit of E. coli consists of three compartments: the A site binds incoming charged tRNAs (tRNAs with their attached specific amino acids). The P site binds charged tRNAs carrying amino acids that have formed bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E site releases dissociated tRNAs so they can be recharged with free amino acids. The ribosome shifts one codon at a time, catalyzing each process that occurs in the three sites. With each step, a charged tRNA enters the complex, the polypeptide becomes one amino acid longer, and an uncharged tRNA departs. The energy for each bond between amino acids is derived from GTP, a molecule similar to ATP ([link]). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid polypeptide could be translated in just 10 seconds.

Termination of translation occurs when a stop codon (UAA, UAG, or UGA) is encountered. When the ribosome encounters the stop codon, the growing polypeptide is released and the ribosome subunits dissociate and leave the mRNA. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

Transcribe a gene and translate it to protein using complementary pairing and the genetic code at this site.

Section Summary

The central dogma describes the flow of genetic information in the cell from genes to mRNA to proteins. Genes are used to make mRNA by the process of transcription mRNA is used to synthesize proteins by the process of translation. The genetic code is the correspondence between the three-nucleotide mRNA codon and an amino acid. The genetic code is “translated” by the tRNA molecules, which associate a specific codon with a specific amino acid. The genetic code is degenerate because 64 triplet codons in mRNA specify only 20 amino acids and three stop codons. This means that more than one codon corresponds to an amino acid. Almost every species on the planet uses the same genetic code.

The players in translation include the mRNA template, ribosomes, tRNAs, and various enzymatic factors. The small ribosomal subunit binds to the mRNA template. Translation begins at the initiating AUG on the mRNA. The formation of bonds occurs between sequential amino acids specified by the mRNA template according to the genetic code. The ribosome accepts charged tRNAs, and as it steps along the mRNA, it catalyzes bonding between the new amino acid and the end of the growing polypeptide. The entire mRNA is translated in three-nucleotide “steps” of the ribosome. When a stop codon is encountered, a release factor binds and dissociates the components and frees the new protein.


Translation / Protein Synthesis

Protein synthesis is the process where cells create proteins.

Explanation:

There are two steps in protein synthesis. They are transcription and translation.

During transcription, mRNA (Messenger RNA) is formed in the nucleus of the cell. After mRNA has been made, it leaves the nucleus and goes to the ribosomes in the cytoplasm, where translation occurs. (DNA never leaves the nucleus.)

During translation, mRNA attaches itself to the ribosome. Then, tRNA (Transfer RNA) reads the mRNA codons (a codon is a sequence of three nucleotides that code for a protein) and attaches amino acids accordingly. This continues until tRNA reaches a stop codon.

Answer:

Codons are three letter genetic words: and the language of genes use 4 letters (=nitrogenous bases). Hence 64 words are there in genetic dictionary, to represent 20 amino acids that the biological organisms use.

Explanation:

And you must note that more than one codon may code for the same amino acid. This is referred to as degeneracy of the code.

For example, three amino acids are coded by any of six different codons, and that alone uses up 18 of the 64 combinations.

Three of the codons are stop codons.

They do not code for any amino acid.

Instead, they act as signals to end the genetic message carried by messenger RNA .

The number of amino acids coded by codons is

#1 " codon" × color(white)(l)2 " amino acids" = color(white)(ll)2 " codons"#
#2 " codons" × 9 " amino acids" = 18 " codons"#
#3 " codons" × 1 " amino acid" = color(white)(X)3 " codons"#
#4 " codons" × 5 " amino acids" = 20 " codons"#
#6 " codons" × 3 " amino acids" = 18 " codons"#
#color(white)(XXXXXXXXXXXXXXXX)3" stop codons"#
#stackrel(—————————————————————————)(color(white)(XXXXXXXXXl)"TOTAL" = 64 " codons")#

Here's a chart that gives the codon assignments for the amino acids.

Answer:

Translation occurs when ribosomes use information from RNA to build proteins.

Explanation:

Translation is the second phase of protein synthesis. It follows transcription, in which the information in DNA is "rewritten" into mRNA. During translation, the mRNA attaches to a ribosome. Transfer RNA (tRNA) molecules then "read" the mRNA code and translate the message into a sequence of amino acids. Every three nucleotides in the mRNA make up one codon, which corresponds to one amino acid in the resulting protein. The ribosome tracks along the mRNA until it reaches a stop codon, signaling the assembly of mRNA and ribosome to break apart.

Below is a stop-motion Vine video that summarizes the steps of translation.

The video below provides a summary of how the processes of transcription and translation occur using the Shockwave tutorial DNA Workshop from PBS.


The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors (Figure 6).

Figure 6: The protein synthesis machinery includes the large and small subunits of the ribosome, mRNA, and tRNA. (credit: modification of work by NIGMS, NIH)

Ribosomes are the part of the cell which reads the information in the mRNA molecule and joins amino acids together in the correct order. In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a very large, complex macromolecule. Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of two subunits that come together for translation, rather like a hamburger bun comes together around the meat (the mRNA). The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule can be simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA “charging,” each tRNA molecule is bonded to its correct amino acid.

Figure 7: Translation begins when a tRNA anticodon recognizes a codon on the mRNA. The large ribosomal subunit joins the small subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release factor into the A site terminates translation and the components dissociate.


Watch the video: Translation (December 2021).