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9.13: Ribozymes - Biology


Until about 20 years ago, all known enzymes were proteins. But then it was discovered that some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules). Catalytic RNA molecules are called ribozymes. Most classes of RNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA) are transcribed as precursors that are larger than the final product. These precursors often contain "head" (5') and "tail" (3') sequences and intron sequences that must be removed to make the final product. Some of the processing steps employ other RNA molecules (always associated with proteins).

Ribonuclease P

Almost all living things synthesize an enzyme — called Ribonuclease P (RNase P) — that cleaves the head (5') end of the precursors of transfer RNA (tRNA) molecules. In bacteria, ribonuclease P is a heterodimer containing a molecule of RNA and one of protein. Separated from each other, the RNA retains its ability to catalyze the cleavage step (although less efficiently than the intact dimer), but the protein alone cannot do the job.

Figure 9.13.1: Crystal structure of a bacterial ribonuclease P holoenzyme in complex with tRNA (yellow), showing metal ions involved in catalysis (pink spheres), PDB: 3Q1R​. (CC BY SA 30; RNAMacGyver).

Group I Introns

Some ribosomal RNA (rRNA) genes, including those in the mitochondrial genome of certain fungi (e.g., yeast), in some chloroplast genomes and in the nuclear genome of some "lower" eukaryotes (e.g., the ciliated protozoan Tetrahymena thermophila and plasmodial slime mold Physarum polycephalum) contain introns that must be spliced out to make the final product.

The splicing reaction is self-contained; that is, the intron — with the help of associated proteins — splices itself out of the precursor RNA. Once excision of the intron and splicing of the adjacent exons are completed, the story is over. In other words, although the action is catalyzed by the RNA, only a single molecule of substrate is involved (unlike protein enzymes that repeatedly catalyze a reaction).

However, synthetic versions of Group I introns made in the laboratory can — in vitro — act repeatedly; that is, like true enzymes. The DNA of some Group I introns includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome. All the Group I introns share a characteristic secondary structure and mode of action that distinguishes them from the next group.

Group II Introns

Some messenger RNA (mRNA) genes in the mitochondrial genome of yeast and other fungi (encoding the proteins cytochrome b and subunits of cytochrome c oxidase) and in some chloroplast genomes also contain self-splicing introns. Because their secondary structure and the details of the splicing reaction differ from the rRNA introns discussed above, these are called Group II introns. The DNA of some Group II introns also includes an open reading frame (ORF) that encodes a transposase-like protein that can make a copy of the intron and insert it elsewhere in the genome.

Spliceosomes

Spliceosomes remove introns and splice the exons of most nuclear genes. They are composed of 5 kinds of small nuclear RNA (snRNA) molecules and over 100 different protein molecules. It is the RNA — not the protein — that catalyzes the splicing reactions. The molecular details of the reactions are similar to those of Group II introns, and this has led to speculation that this splicing machinery evolved from them.

Viroids

Viroids are DNA molecules that infect plant cells as conventional viruses do, but are far smaller (one has only 246 nucleotides). They are naked; that is, they are not encased in a capsi like viruses. Some viroidlike molecules get into the cell as passengers inside a conventional plant virus. These are called virusoids or viroidlike satellite RNAs.

In both cases, the molecules consists of single-stranded RNA whose ends are covalently bonded to form a circle. There are several regions where base-pairing occurs across adjacent portions of the molecule. New viroids and virusoids are synthesized by the host cell as long precursors in which the viroid structure is tandemly repeated. These repeats must be cut out and ligated to form the final product. Most virusoids and at least one viroid are self-splicing; that is, they can cut themselves out of the precursor and ligate their ends without the aid of any host enzymes. Thus they represent another class of ribozyme.

Both viroids and virusoids are responsible for a number of serious diseases of economically important plants, e.g. the coconut palm and chrysanthemums. (The problem is so severe with chrysanthemums that all growers in the U.S. now secure their stock from a few companies that raise the plants in "clean" rooms using stringent precautions to prevent infection by the viroid.)


Catalytic RNA, ribozyme, and its applications in synthetic biology

Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of "RNA" molecules hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic biology, a variety of ribozymes have been developed and engineered. Here, we summarize the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological systems.

Keywords: Activity regulation Catalytic RNA Detecting system Expression control Gene editing Logic circuit Ribozyme Self-cleavage Splicing Synthetic biology.


9.12 Male bias and extreme sex ratios

Figure 9.13 Likelihood of a Male Birth, by Country

Note a clear pattern of increasing male births in South Korea, China, and India, largely associated with increased access to ultrasound and selective-abortion technology. In one study of abortion clinics in India, of 8000 abortions that followed a sex-determination procedure,7,999 were of female fetuses. In another study, not a single one of 250 identified male fetuses were aborted. In the city of Taegu, South Korea, a sex ratio of approximately 1.0 in 1980 became a sex ratio between 1.2 and 1.3 in 1990. That’s roughly 125 males for every 100 females carried to term.

These modern techniques of sex-selection follow a history of neglect or infanticide of female offspring, practices that are well documented in the literature and have led to some dramatically skewed sex ratios. Some villages in India have documented ratios as high as 100:31. When these ratios are extrapolated to populations of tens of millions of people, the inevitable consequence is millions of “missing girls.” The following data summarize this phenomenon of millions of missing girls in Asia.

Figure 9.14 Number of missing females for selected Asian countries, 2001

These data give rise to myriad additional questions: Why is there such a strong bias for sons? Does this bias exist outside of Asia? Do these preferences increase with decreasing fertility, such as during China’s “One Child Policy” of the recent past?

One thing that does seem fairly well documented is that these preferences do not seem to be driven by the mother. Several studies have illustrated that mothers have little expressed preference for sons over daughters, while fathers around the world tend to prefer sons. A study of sex ratios gives us never-ending food for thought!

Read More

Read more here and check out some interactive graphs!

  1. Abrevaya, J. (2009). Are there missing girls in the United States? Evidence from birth data. American Economic Journal: Applied Economics, 1(2), 1–34. https://doi.org/10.1257/app.1.2.1] &crarr
  2. [from Hesketh, T., & Xing, Z. W. (2006). Abnormal sex ratios in human populations: causes and consequences. Proceedings of the National Academy of Sciences of the United States of America, 103(36), 13271–13275. https://doi.org/10.1073/pnas.0602203103, using data from Hudson V.&Den Boer, A.M. (2004) Bare Branches: The Security Implications of Asia’s Surplus Male Population (MIT Press, Cambridge, MA) and Klasen, S. & Wink, C. (2002) Popul. Dev. Rev. 28, 285–312.] &crarr

Telomere Replication

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres, which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 9.11) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Figure 9.11 The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure 9.12) received the Nobel Prize for Medicine and Physiology in 2009.

Figure 9.12 Elizabeth Blackburn, 2009 Nobel Laureate, was the scientist who discovered how telomerase works. (credit: U.S. Embassy, Stockholm, Sweden)

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.


Contents

Before the discovery of ribozymes, enzymes, which are defined as catalytic proteins, [7] were the only known biological catalysts. In 1967, Carl Woese, Francis Crick, and Leslie Orgel were the first to suggest that RNA could act as a catalyst. This idea was based upon the discovery that RNA can form complex secondary structures. [8] These ribozymes were found in the intron of an RNA transcript, which removed itself from the transcript, as well as in the RNA component of the RNase P complex, which is involved in the maturation of pre-tRNAs. In 1989, Thomas R. Cech and Sidney Altman shared the Nobel Prize in chemistry for their "discovery of catalytic properties of RNA." [9] The term ribozyme was first introduced by Kelly Kruger et al. in 1982 in a paper published in Cell. [1]

It had been a firmly established belief in biology that catalysis was reserved for proteins. However, the idea of RNA catalysis is motivated in part by the old question regarding the origin of life: Which comes first, enzymes that do the work of the cell or nucleic acids that carry the information required to produce the enzymes? The concept of "ribonucleic acids as catalysts" circumvents this problem. RNA, in essence, can be both the chicken and the egg. [10]

In the 1980s Thomas Cech, at the University of Colorado at Boulder, was studying the excision of introns in a ribosomal RNA gene in Tetrahymena thermophila. While trying to purify the enzyme responsible for the splicing reaction, he found that the intron could be spliced out in the absence of any added cell extract. As much as they tried, Cech and his colleagues could not identify any protein associated with the splicing reaction. After much work, Cech proposed that the intron sequence portion of the RNA could break and reform phosphodiester bonds. At about the same time, Sidney Altman, a professor at Yale University, was studying the way tRNA molecules are processed in the cell when he and his colleagues isolated an enzyme called RNase-P, which is responsible for conversion of a precursor tRNA into the active tRNA. Much to their surprise, they found that RNase-P contained RNA in addition to protein and that RNA was an essential component of the active enzyme. This was such a foreign idea that they had difficulty publishing their findings. The following year, Altman demonstrated that RNA can act as a catalyst by showing that the RNase-P RNA subunit could catalyze the cleavage of precursor tRNA into active tRNA in the absence of any protein component.

Since Cech's and Altman's discovery, other investigators have discovered other examples of self-cleaving RNA or catalytic RNA molecules. Many ribozymes have either a hairpin – or hammerhead – shaped active center and a unique secondary structure that allows them to cleave other RNA molecules at specific sequences. It is now possible to make ribozymes that will specifically cleave any RNA molecule. These RNA catalysts may have pharmaceutical applications. For example, a ribozyme has been designed to cleave the RNA of HIV. If such a ribozyme were made by a cell, all incoming virus particles would have their RNA genome cleaved by the ribozyme, which would prevent infection.

Despite having only four choices for each monomer unit (nucleotides), compared to 20 amino acid side chains found in proteins, ribozymes have diverse structures and mechanisms. In many cases they are able to mimic the mechanism used by their protein counterparts. For example, in self cleaving ribozyme RNAs, an in-line SN2 reaction is carried out using the 2’ hydroxyl group as a nucleophile attacking the bridging phosphate and causing 5’ oxygen of the N+1 base to act as a leaving group . In comparison, RNase A, a protein that catalyzes the same reaction, uses a coordinating histidine and lysine to act as a base to attack the phosphate backbone. [2] [ clarification needed ]

Like many protein enzymes metal binding is also critical to the function of many ribozymes. [11] Often these interactions use both the phosphate backbone and the base of the nucleotide, causing drastic conformational changes. [12] There are two mechanism classes for the cleavage of phosphodiester backbone in the presence of metal. In the first mechanism, the internal 2’- OH group attacks phosphorus center in a SN2 mechanism. Metal ions promote this reaction by first coordinating the phosphate oxygen and later stabling the oxyanion. The second mechanism also follows a SN2 displacement, but the nucleophile comes from water or exogenous hydroxyl groups rather than RNA itself. The smallest ribozyme is UUU, which can promote the cleavage between G and A of the GAAA tetranucleotide via the first mechanism in the presence of Mn 2+ . The reason why this trinucleotide rather than the complementary tetramer catalyze this reaction may be because the UUU-AAA pairing is the weakest and most flexible trinucleotides among the 64 conformations, which provides the binding site for Mn 2+ . [13]

Phosphoryl transfer can also be catalyzed without metal ions. For example, pancreatic ribonuclease A and hepatitis delta virus(HDV) ribozymes can catalyze the cleavage of RNA backbone through acid-base catalysis without metal ions. [14] [15] Hairpin ribozyme can also catalyze the self-cleavage of RNA without metal ions but the mechanism is still unclear. [15]

Ribozyme can also catalyze the formation of peptide bond between adjacent amino acid by lowering the activation entropy. [14]

Although ribozymes are quite rare in most cells, their roles are sometimes essential to life. For example, the functional part of the ribosome, the biological machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg 2+ as cofactors. [16] In a model system, there is no requirement for divalent cations in a five-nucleotide RNA catalyzing trans-phenylalanation of a four-nucleotide substrate with 3 base pairs complementary with the catalyst, where the catalyst/substrate were devised by truncation of the C3 ribozyme. [17]

The best-studied ribozymes are probably those that cut themselves or other RNAs, as in the original discovery by Cech [18] and Altman. [19] However, ribozymes can be designed to catalyze a range of reactions (see below), many of which may occur in life but have not been discovered in cells. [20]

RNA may catalyze folding of the pathological protein conformation of a prion in a manner similar to that of a chaperonin. [21]

RNA can also act as a hereditary molecule, which encouraged Walter Gilbert to propose that in the distant past, the cell used RNA as both the genetic material and the structural and catalytic molecule rather than dividing these functions between DNA and protein as they are today this hypothesis is known as the "RNA world hypothesis" of the origin of life. [22] Since nucleotides and RNA and thus ribozymes can arise by inorganic chemicals, they are candidates for the first enzymes, and in fact, the first "replicators", i.e. information-containing macro-molecules that replicate themselves. An example of a self-replicating ribozyme that ligates two substrates to generate an exact copy of itself was described in 2002. [23] The discovery of catalytic activity of RNA solved the "chicken and egg" paradox of the origin of life, solving the problem of origin of peptide and nucleic acid central dogma. According to this scenario, in earliest life all enzymatic activity and genetic information encoding was done by one molecule, the RNA.

Since the discovery of ribozymes that exist in living organisms, there has been interest in the study of new synthetic ribozymes made in the laboratory. For example, artificially-produced self-cleaving RNAs that have good enzymatic activity have been produced. Tang and Breaker [24] isolated self-cleaving RNAs by in vitro selection of RNAs originating from random-sequence RNAs. Some of the synthetic ribozymes that were produced had novel structures, while some were similar to the naturally occurring hammerhead ribozyme. In 2015, researchers at Northwestern University and the University of Illinois at Chicago have engineered a tethered ribosome that works nearly as well as the authentic cellular component that produces all the proteins and enzymes within the cell. Called Ribosome-T, or Ribo-T, the artificial ribosome was created by Michael Jewett and Alexander Mankin. [25] The techniques used to create artificial ribozymes involve directed evolution. This approach takes advantage of RNA's dual nature as both a catalyst and an informational polymer, making it easy for an investigator to produce vast populations of RNA catalysts using polymerase enzymes. The ribozymes are mutated by reverse transcribing them with reverse transcriptase into various cDNA and amplified with error-prone PCR. The selection parameters in these experiments often differ. One approach for selecting a ligase ribozyme involves using biotin tags, which are covalently linked to the substrate. If a molecule possesses the desired ligase activity, a streptavidin matrix can be used to recover the active molecules.

Lincoln and Joyce developed an RNA enzyme system capable of self replication in about an hour. By utilizing molecular competition (in vitro evolution) of a candidate RNAmixture, a pair of ribozymes emerged, in which each synthesizes the other by joining synthetic oligonucleotides, with no protein present. [26]

Although not true catalysts, the creation of artificial self-cleaving riboswitches, termed aptazymes, has also been an active area of research. Riboswitches are regulatory RNA motifs that change their structure in response to a small molecule ligand to regulate translation. While there are many known natural riboswitches that bind a wide array of metabolites and other small organic molecules, only one ribozyme based on a riboswitch has been described, glmS. [27] Early work in characterizing self-cleaving riboswitches was focused on using theophylline as the ligand. In these studies an RNA hairpin is formed which blocks the ribosome binding site, thus inhibiting translation. In the presence of the ligand, in these cases theophylline, the regulatory RNA region is cleaved off, allowing the ribosome to bind and translate the target gene. Much of this RNA engineering work was based on rational design and previously determined RNA structures rather than directed evolution as in the above examples. More recent work has broadened the ligands used in ribozyme riboswitches to include thymine pyrophosphate (2). Fluorescence-activated cell sorting has also been used to engineering aptazymes. [28]

RNA polymerase ribozyme Edit

Modern life, based largely on DNA as the genetic material, is thought to have descended from RNA-based organisms in an earlier RNA world. RNA life would have depended on an RNA-dependent RNA polymerase ribozyme to copy functional RNA molecules, including copying the polymerase itself. Tjhung et al. [29] have obtained an RNA polymerase ribozyme by in vitro evolution that has an unprecedented level of activity in copying complex RNA molecules. However, this ribozyme is unable to copy itself and its RNA products have a high mutation rate. Nevertheless, progress continues to be made towards the goal of obtaining, by in vitro evolution, an accurate, efficient self-reproducing RNA polymerase ribozyme in order to improve understanding of the early evolution of life.

Samanta and Joyce [30] found that a highly evolved RNA polymerase ribozyme was able of function as a reverse transcriptase, that is, it can synthesize a DNA copy using an RNA template. Such an activity is considered to have been crucial for the transition from RNA to DNA genomes during the early history of life on earth. Reverse transcription capability could have arisen as a secondary function of an early RNA dependent RNA polymerase ribozyme.

Ribozymes have been proposed and developed for the treatment of disease through gene therapy (3). One major challenge of using RNA based enzymes as a therapeutic is the short half-life of the catalytic RNA molecules in the body. To combat this, the 2’ position on the ribose is modified to improve RNA stability. One area of ribozyme gene therapy has been the inhibition of RNA-based viruses.

A type of synthetic ribozyme directed against HIV RNA called gene shears has been developed and has entered clinical testing for HIV infection. [31] [32]

Similarly, ribozymes have been designed to target the hepatitis C virus RNA, SARS coronavirus (SARS-CoV), [33] Adenovirus [33] and influenza A and B virus RNA. [34] [35] [36] [33] The ribozyme is able to cleave the conserved regions of the virus’s genome which has been shown to reduce the virus in mammalian cell culture. [37] Despite these efforts by researchers, these projects have remained in the preclinical stage.


Ribozyme and RNA World Hypothesis | Cell Biology

According to this hypothesis primordial cells lacking protein synthesis use RNA both as the repository of genetic information and as enzymes that catalyse metabolism. Enzymatic catalysis which was earlier thought to be the exclusive domain of proteins, but is now established that RNA molecules can cut, splice and assemble themselves without any outside help.

These RNA molecules working as enzymes were called ribozymes. Cech (1982) while working on spli­cing mechanism of RNA molecules during pro­cessing of hnRNA into mRNA or precursor rRNA into rRNA in Tetrahymena, found that removal of proteinaceous enzymes one by one from a cell-free system did not alter the splicing ability of pre-ribosomal RNA.

Later in 1984, Altman using ribonuclease Pi (RNA + Protein) in E. coli demonstrated that catalytic activity of the enzyme resided only in RNA subunit and protein only helped RNA to perform the enzymatic func­tion. Self-cleavage of viroid RNAs or virusoids (satellite RNAs) and catalytic activity of RNase are other examples of ribozymes.

Subsequently, it was shown that ribozymes may also bring about cleavage of DNA molecules and selective­ly amplify RNA molecules.

The discovery of RNA molecules working as enzymes has also changed our idea about origin of cell. It is now believed that four billion years ago, earth was an ‘RNA world’ in which RNA molecules carried out all the processes of life without the help of either protein or DNA.

Early living cells were RNA life-forms. A number of facts suggest that heredi­ty was originally based on RNA rather than on DNA. Due to difficulty in synthesizing nucleotides in ‘primordial soup experiments’ it has been speculated that there was an even ear­lier, although chemically related, precursor to RNA that was a carrier of genetic information.

Arguments in favour are:

1. RNA polymerization and replication can take place in vitro in a purely chemical sys­tem based on nucleotides.

2. The genome of certain viruses is based on RNA.

3. RNA is more complex than stable double stranded DNA because single stranded RNA can fold up in different three dimensional configurations (e.g., tRNA).

4. RNA can have a catalytic effect, so called ribozymes catalyse cutting and splicing of other RNA molecules. RNA molecule even catalyses the polymerization and replication of RNA.

5. The universal and central importance of RNA in protein synthesis.

6. Synthesis of deoxyribose of DNA takes place via ribose of RNA indicating that DNA rep­resents a further and later development of RNA.

7. Nucleosides function as co-enzymes to many enzymes, e.g., ATP. This role of nucleotides can be incorporated as a relict from the RNA world.

RNA molecules were the most important entities in the primitive structural organisation tRNA and rRNA possess a folded three-dimensional structure that may have participated in making informational proteins. Primitive ribosomes were without any protein and capable of making peptides with the help of tRNA that could bind with rRNA without the involvement of enzyme system.

tRNA molecule could link with a given amino acid at the anticodon site through non-covalent linkage indicating enzyme­ like role of primitive tRNA and rRNA.

Linking another RNA strand with tRNA and rRNA for error-free amino acid sequence resulted in the development of template system with the forma­tion of first mRNA. The formation of catalytic protein may have followed the synthesis of nucleoprotein com­plexes. Later developed the enzyme system catalysing the formation of peptide bonds for efficient translation.

As nucleic acids can serve as template in the absence of enzymes and pro­teins, a complementary nucleic acid may have been formed in the presence of a suitable con­densing agent. This led to the formation of DNA which had the self-replicating property and permanent information-storage capacity.

It is possible to study RNA world in the labo­ratory. RNA isolated from virus (Qβ) replicates in a test tube in the presence of an RNA replicase, and four nucleoside triphosphates (ATP, UTP, CTP and GTP). Such RNA culture could at intervals be transferred to fresh medium so that its development could be followed over longer time periods (Fig. 2.7).

Further experiments (Eigen) showed that without the help of RNA primer, in a system containing RNA replicase enzyme, four nucleoside triphos­phates and Mg ++ , RNA strands appeared and replicated.

The RNA molecules formed con­tained about a hundreds of nucleotides and due to internal base pairing they have a hairpin-like shape (Fig. 2.8) termed as quasi-species. Though the replication of RNA depends on the presence of the complex enzyme RNA replicase but attempts have been made by Orgel to make replication of short RNA molecules in enzyme- free system. Thouston et al. have produced an

RNA molecule that catalyses the replication of RNA (ribozyme). The RNA world hypothesis pre­sents a real advance in attempts to understand the origin of cell.

Proto-Cell in RNA World:

RNA replicase is a key component of proto-cell, that can act both as a template for the storage and transmission of genetic information, and as an RNA polymerase that can replicate its own sequence. Simple proto-cell consists of an RNA replicase replicating inside a replicating membrane vesicle (Fig. 2.9).

Both these compo­nents are self-assembling as RNA molecules can become encapsulated in vesicles as they form, the proto-cell as a whole could self-assemble.

With compartmentation the replicase is capable of vari­ation and natural selection and then Darwinian evolution. An RNA coded activity (e.g., ribozyme) is needed that inputs an advantage in growth of membrane component. A single cell with inter­dependent genome and membrane would be a sustainable, autonomously replicating system.


The biochemistry of programmed cell death

Programmed cell death (PCD) is involved in the removal of superfluous and damaged cells in most organ systems. The induction phase of PCD or apoptosis is characterized by an extreme heterogeneity of potential PCD-triggering signal transduction pathways. During the subsequent effector phase, the numerous PCD-indueing stimuli converge into a few stereotypical pathways and cells pass a point of no return, thus becoming irreversibly committed to death. It is only during the successive degradation phase that vital structures and functions are destroyed, giving rise to the full-blown phenotype of PCD. Evidence is accumulating that cytoplasmic structures, including mitochondria, participate in the critical effector stage and that alterations commonly considered to define PCD (apoptotic morphology of the nucleus and regular, oligonucleosomal chromatin fragmentation) have to be ascribed to the late degradation phase. The decision as to whether a cell will undergo PCD or not may be expected to be regulated by “switches” that, once activated, trigger self-am- plificatory metabolic pathways. One of these switches may reside in a perturbation of mitochondrial function. Thus, a decrease in mitochondrial transmem- brane potential, followed by mitochondrial uncoupling and generation of reactive oxygen species, precedes nuclear alterations. It appears that molecules that participate in apoptotic decisionmaking also exert functions that are vital for normal cell proliferation and intermediate metabolism.—Kroemer, G., Petit, P., Zamzami, N., Vayssière, J.-L., Mignotte, B. The biochemistry of programmed cell death. FASEB J. 9, 1277-1287 (1995)


SB2244 Molecular Biology #9 - #13

If a ddNTP analog is present, it can be combined into the growing DNA chain which could terminate the chain at that point.

It competes with dNTP, so that ddNTPs are randomly incorporated into the growing strand.

This will produce a set of DNAs of different lengths complementary to the template DNA.

The lengths can be used to determine the position of each nucleotides in the growing chain.

The sets of DNA of different lengths can be separated using PAGE (one lane for ddATP, one lane for ddGTP, etc) which are detected by a label (radioactive/fluorescent) incorporated into the primer or into one of the dNTP.

E.g. If a DNA has T residues at positions 2, 5, 13, 16, etc. this can be converted into a set of DNAs of length n+2, 5, 13, 16, etc. (which can be measured by denaturing polyacrylamide gel electrophoresis).

It is also can be used for the detection of differentially represented repetitive DNA.

It involved the hybridization of isolated DNA (cDNA from a related organism with DNA Label probe) from each of the E.coli/phage.

But it is important that the temperature is not so high that it washes the probe off of clones that contain sequences that are similar or identical to the probe itself.

Lacks 3' to 5' exonuclease proofreading activity.

- Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs these can hybridize inappropriately on the template, or generate complex structures within the primer

- Aim for a GC content of 40 to 60%

- If possible, the 3' end of the primer should end in GC bases i.e. GC clamp to enhance annealing of the end which will be extended.

- Inverted repeat sequences should be avoided to prevent formation of secondary structure in the primer which may prevent hybridization to template.

- Minimize primer complementarity to prevent hybridization between primers i.e. primer dimers

- Primer pairs should have similar Tm values i.e. within 2°C

The technique relies on the ability of A and T on different DNA fragments to hybridize and become ligated together with the presence of ligase.

PCR products are usually amplified using Taq polymerase which preferentially adds an adenine to the 3' end of the product.

Products are detected as they are made.

It is less time consuming than conventional PCR - no end product analysis by electrophoresis.

Characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers.

Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

The amount of PCR product is determined by measuring the level of fluorescent probe attached to a reporter oligonucleotide complementary to the DNA segment that is being amplified i.e. High level of unattached fluorescent probes = PCR product is high.

The looping is facilitated in some cases by proteins called architectural regulators that bind to intervening sites and facilitate the looping of the DNA.

Most of the eukaryotic systems involve protein activators.

The actual interaction between the activators and the RNA polymerase at the promoter is often mediated by intermediary proteins called coactivators.

If lactose is present, the repressor dissociates from the operator. However, if glucose is also available, low cAMP levels prevent CRP-cAMP formation and DNA binding. RNA polymerase may occasionally bind and initate transcription, resulting in a very low level of lac gene transcription.

When lactose is present and glucose levels are low, cAMP levels rise. The CRP-cAMP complex forms and facilitates robust binding of RNA polymerase to the lac promoter and high level or transcription.

a. It has only two states, it is either fully on or fully off it is not a dimmer switch. The target gene or operon is either fully expressed or not expressed at all. It cannot be expressed at an intermediate level.

e.g. Receptors for estrogen, progesterone.

e.g. The thyroid hormone receptor

RNA polymerase and the transcription bubble move from left to right along the DNA, facilitating RNA synthesis.

The DNA is unwound ahead and rewound behind as RNA is transcribed.

As the DNA is rewound, the RNA-DNA hybrid is displaced and the RNA strand is extruded.

Movement of an RNA polymerase along DNA tends to create positive supercoils i.e. overwound DNA, ahead of the transcription bubble and negative supercoils i.e. underwound DNA, behind it.

The RNA polymerase is in close contact with the DNA ahead of the transcription bubble as well as with the separated DNA strands and the RNA within and immediately behind the bubble.

A channel in the protein funnels new NTPs to the polymerase active site.

DNA fragments thought to contain sequences recognized by a DNA-binding protein and radiolabel one end of one strand were isolated. Chemical or enzymatic reagents were used to introduce random breaks in the DNA fragments.

2. A transcription bubble unwound to form an open complex.

3. Transcription is initiated.

4. Promoter clearance is followed by elongation.

5. Elongation continues. σ70 subunit dissociates, and is replaced by NusA protein.

It has RNA components called small nuclear RNAs (snRNAs, U1 - U6, - 200 bp).

snRNAs together with 6-10 proteins form a complex called small nuclear ribonucleoprotein particles (snRNPs).

Self-splicing RNAs occur also in mitochondria, chloroplasts and bacteria.

In the thyroid, this exon is retained.

The primary transcript can be spliced in different ways to produce mRNAs, which then give rise to variant proteins.

Some of the splicing patterns are specific for certain types of cells.

2. Cleavage liberate precursors of rRNAs and tRNAs. Cleavage are carried out by the enzymes RNase III, RNase P and RNase E.

2. A series of enzymatic cleavages of the 45S precursor produces 18S, 5.8S, and 28S rRNAs, and the ribosomal subunits gradually take shape with the assembling ribosomal proteins.

3. The cleavage reactions and all the modifications require small nucleolar RNAs (snoRNAs) found in protein complexes (snoRNPs) in the nucleolus that are similar to spliceosomes.

The ends are processed first, the 5' end before the 3' end.

CCA is then added to the 3' end, a necessary step in processing eukaryotic tRNAs and those bacterial tRNAs that lack this sequence in the primary transcript.

While the ends are being processed, specific bases in the rest of the transcript are modified.

its processing is mediated by two endoribonucleases in the RNase III family, Drosha and Dicer.

First, in the nucleus, the pri-miRNA is reduced to a 70 to 80 nucleotide precursor miRNA (pre-miRNA) by a protein complex including Drosha and another protein, DGCR8.

The pre-miRNA is then exported to the cytoplasm in a complex with a protein called exportin-5 and the Ran GTPase.

In the cytoplasm, Ran hydrolyzes
the GTP, then exportin-5 protein and the pre-miRNA are released.

The Ran-GDP and exportin-5 proteins are transported back into the nucleus.

The pre-miRNA is acted on by Dicer to produce the nearly mature miRNA
paired with a short RNA complement.

The complement is removed by an
RNA helicase, and the mature miRNA is incorporated into protein complexes, such as the RNA-induced silencing complex (RISC), which then
bind a target mRNA.

If the complementarity between miRNA and its target is nearly perfect, the target mRNA is cleaved.


Watch the video: Ribozyme (January 2022).