What causes the complementary copy of an RNA molecule to separate?

What causes the complementary copy of an RNA molecule to separate?

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I have recently read an article which explains that, in the RNA World hypothesis, an RNA molecule gets 'scanned' by nucleic acid, catalysed by a different specifically-folded RNA molecule, to arrange itself complimentarily to the original molecule. However, what causes the new, complementary molecule to detach from the original molecule? I always hear people say that this complementary base pairing allows the RNA molecule to reproduce, but I never quite understood: how? If complementary bases tend to bind together, shouldn't the newly formed structure then be stable? What causes the actual reproduction?

First, I want to point out that the RNA world hypothesis is just that - an hypothesis. While it has been shown that it's possible for certain RNA molecules to make copies of itself, this is not a 'normal' function of any RNA.

Edit - to give a clearer answer to the question itself:

After replication of an RNA molecule it may form a stable structure together with its template or it may dissociate. Due to the various possibilities and complexity of RNA structures its almost impossible to predict this, since both the (exact) sequence itself and environmental conditions especially temperature are very important.

More background from the unedited answer:

Whether two complementary strands or RNA (or DNA) bind together is not something that can always be easily answered. For DNA of a given length (in a given environment / buffer) one can more or less predict the temperature at which complementary strands will separate (often called melting temperature), because DNA forms relatively stable helices. RNA, however, often forms complicated 3-dimensional structures, often involving self-complementary and is not restricted to the typical helices seen in DNA. Trying to predict the resulting 3D structure of RNA molecules is still an ongoing field of research. Some of these structure are quite stable (e.g. in tRNA) but in other cases they can also be very dynamic and change quickly. In the end it always depends on the RNA sequence, temperature and many other environmental factors.

Intro to RNA Biology: Cartoon Edition

DNA contains the instructions for how to build everything in our bodies. Each of your cells contains a copy of your DNA—like a set of the blueprints. Parts of these instructions are carried out as needed in different cells.

Typically, the instructions in the DNA blueprints are followed to build different proteins. Proteins do much of the work needed to keep the body functioning. At any given moment, one cell might be following the instructions for how to make an immune protein to defend the body from invaders like viruses while another is making a protein that helps the stomach digest food.

Jennifer Cook-Chrysos/Whitehead Institute

Generally, DNA is kept inside of the nucleus, where there is a central blueprint library for the whole cell. However, proteins get built in a different part of the cell. How do the instructions for the proteins get transmitted from the DNA blueprints to where the proteins are actually built? They are carried in the form of messenger RNA, or mRNA. Each mRNA contains a copy of a specific blueprint from the larger DNA library. It carries this blueprint out into the cell, where proteins are made. (mRNAs are not the only kind of RNA. There are lots of other types that do other things, and we’ll say more about them later on!)

Jennifer Cook-Chrysos/Whitehead Institute

The mRNA is created in a process called transcription. An enzyme called RNA polymerase travels along the DNA strand, bringing in complementary RNA building blocks to create a “transcript” of the information contained in the DNA. The resulting molecule is an RNA strand with a mission: to convey its message to the part of the cell that creates proteins.

Jennifer Cook-Chrysos/Whitehead Institute

Jennifer Cook-Chrysos/Whitehead Institute

Once the transcript is created, it must undergo a few modifications to be ready for translation (the process by which the contents of the mRNA are read by a molecular machine called a ribosome and translated into a sequence of protein building blocks called amino acids.)

Jennifer Cook-Chrysos/Whitehead Institute

Jennifer Cook-Chrysos/Whitehead Institute

How is mRNA regulated?

Cells have evolved several ways to regulate which proteins are made and when. mRNAs will naturally degrade over time, with most having a relatively short life span: mRNA molecules in mammalian cells can stick around for anywhere from a few minutes to a few days before they are degraded. One way that cells regulate how much of a certain protein is made at a given time is by controlling how long an mRNA remains intact. As long as an mRNA is intact, it can be translated over and over to produce more proteins.

Some mRNAs last for as much as one thousand times longer than others. One of the main regulators of how long mRNAs remain intact is a different type of RNA, called a miroRNA (miRNA).

Small but powerful

MicroRNAs are one of the many types of RNAs that don’t code for proteins. These tiny RNAs, which are only 22 building blocks long, don’t get translated in the ribosome like messenger RNAs. Instead, they target and bind to sequences in specific mRNAs and can block the mRNA from being translated.

Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key-residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

Slipping past the proofreader

In the early 2000s, when experts believed human coronavirus infection caused nothing worse than the common cold, Mark Denison struggled to land adequate federal grants to support his lab at the Vanderbilt University Medical Center.

A virologist and clinician, Denison had been studying coronaviruses since 1984, when only two of the seven coronaviruses currently known to cause disease in humans were known. Those two, along with several other coronaviruses, cause colds.

Denison initially found the virus he studied, which affects only mice, interesting because it leads to a mouse version of multiple sclerosis. Along the way, he became interested in how the virus replicated &mdash but persuading funders to support his work was a real challenge.

In early 2003, he and his wife, Laura, were on vacation in Florida having a difficult conversation. &ldquoI think the work is important. I think the models are important,&rdquo he recalled saying. &ldquo(But) I don&rsquot know if I can maintain a career.&rdquo

That was the day he learned about the pathogen behind the deadly respiratory disease SARS, which had been making headlines.

&ldquoI&rsquom literally on the beach with my wife, and someone came down from the hotel and told me that I had a phone call,&rdquo he said. The call was from a colleague, sharing the news that the pathogen responsible for the disease had been identified, and it was a coronavirus.

Severe acute respiratory syndrome swept from southern China into 26 countries in 2002 and 2003, killing about 10% of the roughly 8,000 people it infected. Although the outbreak was contained, coronaviruses were recognized suddenly as a serious problem of potentially pandemic proportion.

Almost two decades later, the work done in Denison&rsquos lab has turned out to be instrumental in developing a molecule, called remdesivir, that entered large-scale clinical trials just weeks after SARS-CoV-2, the coronavirus that causes COVID-19, was identified.

The molecule, it seems, can overcome coronaviruses&rsquo superpower: their genome-proofreading ability. This ability isn&rsquot found in other RNA viruses, and it makes coronaviruses resistant to a majority of drugs used against other RNA viruses.

Researchers expect to learn in the next few weeks whether remdesivir is an effective treatment for patients with COVID-19. In the meantime, Denison&rsquos lab has continued to work to identify other molecules that might bypass viral resistance.

&ldquoThis is a really key question for us: Is this a new class of drug that can allow us to better design more drugs that can bypass the proofreading function and inhibit the virus?&rdquo said Denison, who now directs the pediatric infectious diseases division at Vanderbilt.

Not your average RNA viruses

Like many viruses that cause human disease, coronaviruses have a genome composed of RNA.

When the virus behind SARS was identified, one of the many experimental treatments clinicians tried using was a molecule called ribavirin. At the time, ribavirin was the first-line drug for many RNA viruses.

Ribavirin targets a viral protein called the RNA-dependent RNA polymerase, or RdRp, which is responsible for replicating the coronavirus genome.

Craig Cameron is a virologist at the University of North Carolina at Chapel Hill who studies RdRp molecular mechanisms in picornaviruses. &ldquo(RdRp) is a very well-validated drug target,&rdquo Cameron said. &ldquoAnd it is one of those targets that actually have the potential of having pan-viral antiviral activity.&rdquo

Ribavirin belongs to a class of antiviral drugs called nucleotide or nucleoside analogs.

Exactly how ribavirin works seems to vary from one virus to another, making it a good illustration of the many possible modes of action of nucleotide analogs. Some work by blocking the viral polymerase, terminating a growing strand of RNA. Some are mutagens, which slip into the growing viral genome, letting the polymerase continue, but introduce some molecular ambiguity to the next round of replication that causes a cascade of errors in later generations. Some block metabolic enzymes, preventing the synthesis or processing of real ribonucleotides and thereby slowing down replication.

A systematic review of data from 30 clinical trials, conducted after the SARS epidemic, showed no conclusive benefit of ribavirin &mdash or any other treatment that was tested &mdash and some evidence that ribavirin had done patients harm.

Ribavirin was the first example. Since then, most nucleotide analogs tried against coronaviruses that cause SARS and Middle East respiratory syndrome, or MERS, have not been effective treatments. Referring to ribavirin and the classic nucleoside analog 5-fluorouracil, which works as a mutagen, Denison said, &ldquoCoronaviruses are completely, entirely resistant to those drugs. You can soak them in it, and they have no effect.&rdquo

Vigorous viral proofreading

Bruno Canard is the principal investigator of a viral replication group at the French National Centre for Scientific Research. Before the SARS outbreak, Canard had focused on the structure&ndashactivity relationships of nucleotide analogs used to treat HIV and other viruses. Like many virologists, he was inspired by the near miss with SARS to start new research programs.

&ldquoWe were surprised that ribavirin was pretty toxic and not very effective (against) coronaviruses,&rdquo Canard said.

Both the CNRS team in Marseille and Denison&rsquos group in Tennessee set out to understand more about the virus that caused SARS, and one of their major questions was why ribavirin, broadly effective against other RNA viruses, had failed against this one.

The answer lay in the virus family&rsquos large genome and how it evolved to protect itself.

&ldquoThe reason (RNA viruses) are thought to be so successful is because their polymerases make mistakes,&rdquo Denison said. &ldquoThey lack the ability to correct mistakes, so they generate mutant swarms of viruses that are ready for adaptation in different environments.&rdquo

Structural biologists describe RNA-dependent RNA polymerases as resembling a cupped hand, with the fingers and thumb protecting the enzyme&rsquos active site. As each new nucleobase in the template strand enters the active site, the polymerase coordinates a new incoming ribonucleotide by matching it against its counterpart in the existing strand. If the fit is right, the enzyme catalyzes a bond formation in the RNA backbone. If the fit is close enough, many viral RNA polymerases will catalyze a bond anyway. An error rate that can be as high as one mistake per 10,000 bases lets those mutant swarms arise. But for coronaviruses, the replication error rate is lower.

Coronaviruses have some of the longest genomes in the RNA viral world. Whereas their closest cousins have genomes averaging 10 kilobases, coronavirus genomes are three times as long. With so much genetic material to copy, if coronaviruses mutated at the same rate as other RNA viruses, they would accumulate so many mutations that they would barely produce any viable progeny.

As researchers in the field came to understand the coronavirus RdRp better, they found that the polymerase by itself could not explain the disconnect. Virologist François Ferron, a staff scientist at CNRS, has worked on coronavirus replication since the SARS outbreak. &ldquoThe viral RNA polymerase is quite loose, meaning it has a tendency to make a lot of mistakes,&rdquo he said. &ldquoMaybe a little bit more than the regular (cellular) RNA polymerase.&rdquo

This led researchers to suspect that coronaviruses might have some way of recognizing and correcting errors.

An international team conducting a study of the SARS viral genome identified a number of potential RNA-processing enzymes based on their homology to other known enzymes. In 2006, Canard&rsquos group worked with members of that international bioinformatics team to show that one of those enzymes, like its homologs, could cleave double-stranded RNA and was required for successful viral replication.

&ldquoThere was a speculation that (coronaviruses) might encode a proofreading function that would allow them to stabilize a big genome, and there was a predicted place in the genome where that might occur,&rdquo Denison explained. &ldquoThat really led us to try the genetic experiments.&rdquo

In 2007, his group found that in viruses lacking the protein encoded at that same location, a protein called nsp14, coronaviruses from a mouse model virus accumulated mutations at a rate similar to other RNA viruses. And strains of the virus without nsp14, they found, were sensitive to ribavirin.

Following up on that work, the French group zeroed in on how nsp14 worked in a test tube. &ldquoWe didn&rsquot work on the virus, like Mark Denison was beautifully doing,&rdquo said Canard. &ldquoWe just concentrated on that enzyme &hellip and we found out that it could actually excise ribavirin.&rdquo

The CNRS team published an enzymology study that confirmed that nsp14 can identify and remove mismatches between bases at the end of a growing copy of viral RNA in 2018 they followed up with confirmation that when ribavirin is incorporated in a growing RNA strand, nsp14 protein can scoop it out of the stalled strand, letting replication resume.

As the researchers worked out the enzymology, a pressing question arose: Did their findings mean that all nucleotide analogs would be useless against coronaviruses?

&ldquoThis question is actually key right now in the development of nucleoside analog inhibitors,&rdquo Canard said.

Anatomy of a molecule: What makes remdesivir unique?

Evading viral proofreading

This is where remdesivir comes in.

&ldquoFrom my perspective, the (remdesivir) story started in 2013,&rdquo Denison said. &ldquoWe had discovered that coronaviruses encode the only known RNA proofreading system &hellip (and) I wanted to test whether there were any nucleosides out there that could be active in the setting of coronavirus proofreading.&rdquo

Denison heard from Cameron about a research collaboration with Gilead Sciences. Cameron was working on understanding the precise mechanism of action of a group of nucleotide analogs the company was using to treat hepatitis C, an RNA virus that infected tens of thousands of people each year.

Before the race to drug hepatitis C, according to Adrian Ray, a medicinal chemist who used to work at Gilead, &ldquoThe nuc space for RNA polymerases and for RNA viruses was really not heavily explored.&rdquo

In the course of trying to beat competitors to the lucrative hepatitis C market, Gilead had developed a large library of RNA-dependent RNA polymerase inhibitors, including the molecule that would come to be known as remdesivir. A compound closely related to remdesivir had made it to early clinical tests for hepatitis C, but had faltered, in part because like remdesivir it needed to be administered by injection. Gilead changed strategies, buying a biotech startup to gain access to the startup&rsquos orally available nucleotide analog, which became a key component of Gilead&rsquos hepatitis C cocktail.

Working with the RNA-dependent RNA polymerase from poliovirus, Cameron had begun work that would show that the molecule was a non-obligate chain terminator, a type of nucleotide analog that the polymerase ought to be able to incorporate into a growing strand and keep going &mdash but could not.

Intrigued, Denison contacted Gilead to ask permission to try that approved drug, called sofosbuvir, against mouse coronaviruses.

&ldquoThat was their world-changing drug that cured hepatitis C,&rdquo Denison said. &ldquoThey weren&rsquot going to let a little-known virologist working on a coronavirus use their drugs.&rdquo But after a series of introductions by Cameron and several discussions, Gilead agreed to let his lab work with a different series of molecules, the ones that had been developed in-house and shelved for hepatitis C. They had shown promising results in early studies as a candidate treatment for other viral infections, including against Ebola virus.

The candidate molecules arrived. Denison and his trainees had no idea what they were. But they went ahead and tested them. What they found was exciting: In mouse cell culture, the drugs could block coronavirus replication.

&ldquoSo we asked our Gilead collaborators, &lsquoWhat is that (compound)?&rsquo They said, &lsquoWe&rsquore not going to tell you, but we&rsquore going to send you 60 prodrugs, chemical modifications of the same compound,&rdquo Denison said.

One of that second batch of molecules proved to be remdesivir. Graduate student Maria Agostini, who had recently joined Denison&rsquos lab, was one of the researchers who worked on understanding its strong activity.

&ldquoWe&rsquove worked with a couple of potent compounds, but remdesivir was really one of the first I had worked with,&rdquo Agostini said. &ldquoWhen you were looking at the cells, you could visually see less evidence of viral replication going on.&rdquo

Whereas cells in her control dishes were visibly infected, suffering bad cytopathic effects, the cells treated with remdesivir after infection survived well. By growing many generations of the virus in cells treated with subtherapeutic concentrations of remdesivir, selecting for mutations that would let the virus evade the drug, Agostini and colleagues Erica Andres and Clint Smith demonstrated that it would take mutations in the viral polymerase to confer resistance to remdesivir &mdash and that those mutant viruses were less able to infect hosts than the wild type.

Gilead supplied the drug free of charge, and the National Institutes of Health funded the researchers through a program aimed at developing treatments for emerging infectious diseases.< /p>

&ldquoThis points out the value of collaborative science,&rdquo Denison said. &ldquoThis was a company that committed to helping us do this when no one was interested in coronaviruses, and a grant mechanism that allows some flexibility in terms of expanding it.&rdquo

Recently, Mathias Götte&rsquos enzymology lab in Canada has looked at how remdesivir works on polymerase enzymes from the coronavirus that causes MERS. Researchers in Götte&rsquos group determined that the compound stops the polymerase, acting as a chain terminator &mdash but not immediately.

Andrea Pruijssers, a virologist who directs the antivirals research program in Denison&rsquos group, said, &ldquo(Remdesivir) somehow evades recognition by the proofreading enzyme.&rdquo

Instead of stopping the polymerase as soon as it is incorporated, remdesivir seems to let the enzyme keep going for a few more cycles but then causes it to stall. Researchers suspect that the molecular stumble may be caused by an unusual structure in the template-copy RNA duplex.

&ldquoThey think that, at that point, the nucleoside analog that has already been incorporated is shielded from the proofreading enzyme,&rdquo Pruijssers said.

In the lab of Denison&rsquos longtime collaborator Ralph Baric at the University of North Carolina, Chapel Hill, researchers found that remdesivir was an effective treatment for mice infected with SARS. Those results were promising enough to advance remdesivir into a study of MERS in monkeys, published in February by researchers at the Rocky Mountain Laboratory of the NIH. The drug showed some benefit in reducing the severity of the illness, provided the monkeys were treated prophylactically.

In terms of drug development, remdesivir &ldquohas sort of met every milestone along the way, from our perspective,&rdquo Denison said.

Similar drug class, different result

When Pruijssers started in the Denison lab in 2017, they had another molecule to investigate, beta-D-N4-hydroxycytidine, or NHC for short, that was being tested at the Emory Institute for Drug Development as a potential broad-spectrum antiviral drug.

Agostini led the first study of that drug as well, showing its activity in tissue culture. In March, researchers from the Baric and Denison labs published a followup in Science Translational Medicine, showing that NHC can block replication in the viruses that cause MERS and SARS &mdash and also the coronavirus that causes COVID-19.

&ldquoIt&rsquos interesting,&rdquo Pruijssers said. &ldquo(NHC) doesn&rsquot act as a chain-terminator. It incorporates into the genome and then causes lethal mutagenesis.&rdquo

Through its ability to base-pair with more than one nucleotide in the complementary strand, NHC introduces a cascade of errors in successive rounds of replication. Eventually, viral progeny don&rsquot have the information they need to make a new virus.

Whereas remdesivir stops the polymerase in its tracks, causing few new mutations, deep sequencing experiments in the few viruses that emerged after NHC treatment showed that the drug causes numerous mutations.

&ldquoWe identified two compounds that structurally fall into the same class of nucleoside analogs but act very differently on coronaviruses,&rdquo Agostini said.

They were particularly encouraged because the work showed that NHC has some therapeutic efficacy in the mouse model of MERS &mdash and that it can block even remdesivir-resistant strains of coronavirus.

In an interview in early March, Pruijssers said NHC was still relatively untested as a therapeutic. &ldquoIt&rsquos hard to develop a mutagen, because the FDA doesn&rsquot usually like the idea of mutation unless it&rsquos for a life-threatening disease.&rdquo

But the pandemic changed things. Emory has partnered with Miami biotechnology company Ridgeback Biotherapeutics to develop the molecule, and Emory has filed an application with the U.S. Food and Drug Administration for permission to begin first-in-human trials to test the molecule&rsquos safety.

Denison described the pivotal role Agostini played: &ldquoIn five years of graduate school, Maria tested and developed the detailed in vitro analysis on two potential drugs to treat this pandemic coronavirus &mdash and got them all the way through that in vitro preclinical development.&rdquo

It&rsquos a remarkable and highly unusual feat. Drug development is known for its high failure rate. Of course, many drug candidates that appear promising in preclinical studies falter in large human trials.

Facing the pandemic

After arguing for a decade that the world must be ready for a pandemic, Denison said in early March, it was strange to be facing it.

&ldquoIt&rsquos really weird that we worked on this for the past six years, and the drugs were just getting through, and they were just ready to go,&rdquo he said. &ldquoWe&rsquoll see what the outcome is.&rdquo

He has some concerns about how to interpret data from clinical trials testing how well remdesivir works for COVID-19 in humans, the first of which are expected to be released this month as a trial at the China&ndashJapan Friendship Hospital in Wuhan concludes. (Other trials, sponsored by Gilead, the NIH and the World Health Organization, launched later.)

First, animal data from other coronaviruses suggest that the drug is most effective when it&rsquos delivered prophylactically, after the animals are exposed but before they begin to develop symptoms. In the context of a global pandemic, this would be difficult to achieve in humans. At some point in the course of infection, an extremely strong immune response begins to do more harm than the virus at that point, Denison said, it may be too late for an antiviral to help.

Second, the data from the first few trials are likely to be nuanced and require careful interpretation &mdash which Denison worries public discourse is not well prepared for. Early human trials of remdesivir&rsquos sister compound in hepatitis patients showed dramatic differences among individuals in antiviral response, and remdesivir itself showed limited benefit compared to other candidate therapies in a clinical trial in Ebola patients. &ldquoPeople tend to have a winner or loser mentality,&rdquo he said. &ldquoThey&rsquove called remdesivir &lsquothat failed Ebola drug,&rsquo right? It didn&rsquot fail in the Ebola trial it just wasn&rsquot advanced because it didn&rsquot show as much benefit as the other two compounds.&rdquo

Whatever the outcome of clinical trials of remdesivir and NHC, Denison said he hopes this crisis will underline the importance of funding research into potentially pandemic viruses before outbreaks begin.

&ldquoTrying to maintain basic investigations and drug development against something that&rsquos a high, high, high impact but low, low, low probability is really hard in our world. Really hard,&rdquo he said. &ldquoWe&rsquove just never given up on this idea that we had to have these things ready and have them in the bucket.&rdquo

The Reverse Transcriptase Ripple

In 1970, two laboratories demonstrated biochemically that RNA could be used as a template for DNA. Howard Temin had argued that the RNA of some viruses must be copied into DNA in certain cell types in order to explain the ‘transformation’ of normal cells to cancer cells by these viruses. Detecting the enzyme responsible for this RNA-dependent DNA synthesis was an important piece of evidence that convinced many former sceptics that his theory was valid. For many, the new results seemed consistent with the predictions of the central dogma, but for others, these findings were seen as a challenge to it. One writer went so far as to suggest that existence of ‘inverted transcription’ (copying RNA to make DNA) meant that the entire central dogma needed to be re-examined. In retrospect it does seem a bit strange that this (anonymous) writer did not bother to reread the original formulation before calling for it to be discarded! Crick responded to the challenge and a few weeks later published an expanded view of the central dogma. He reiterated that information transfer from RNA to RNA, from RNA to DNA, or perhaps even from DNA to protein (directly, without an RNA intermediate) were all ‘special transfers’ which might occur in certain cell types. As such, they were perfectly consistent both with the sequence hypothesis and with the flow of information among nucleic acids or from nucleic acids to proteins. Thus the newly discovered ‘reverse transcriptases’, as the RNA-dependent DNA polymerases are now called, did not disturb the central dogma, despite the initial ‘ripple’. However, Crick also pointed to three ‘unknown transfers’, from protein to either protein, RNA or DNA. Such transfers, if shown to exist, would require a radical reformulation of molecular principles. If the gene products (proteins) could alter genes (DNA sequences), the way would be paved for the inheritance of acquired characteristics. In 1970, Crick felt that the available evidence was still insufficient to conclude that the central dogma was certain to be correct, although he maintained that it was likely to remain useful. More recently, it was found that under certain circumstances, the DNA sequences of an organism became altered in what appeared to be a directed way in response to environmental stimuli. In addition to expanding our understanding of the origin of mutations, the experiments raised the possibility that ‘advantageous’ proteins might cause ‘advantageous’ mutations. Thus analysis of the origin of ‘adaptive’ mutations is crucial to evaluating the validity of the central dogma, since these raised the possibility that information might flow from proteins to nucleic acids. See also Retroviral Replication, Baltimore, David, and Temin, Howard Martin

'RNA World'

RNA's versatility in function and form helped inspire the idea known as the "RNA world" hypothesis.

Organisms rely on an astoundingly complicated system of DNA, RNA and protein to transmit hereditary information, and scientists have long wondered how this system could have arisen in early life-forms. RNA offers a logical answer, He said: This molecule can both store genetic information and catalyze reactions, suggesting that early, simple organisms could have relied solely on RNA.

"It's a hybrid," He said. "So it makes perfect sense as a start."

Additionally, He said, RNA's sugar base, ribose, always appears first in organisms, as it's easier to make. Deoxyribose then gets created from ribose. "So that implies in life, you have the ribose, the RNA first, and then the DNA comes later," He said.

From that simpler RNA start, more-complex life could arise, evolving the stabler DNA to serve as a long-term library and developing protein as a more efficient catalyst.

Functions of RNA Polymerase

Traditionally, the central dogma of molecular biology has looked at RNA as a messenger molecule, that exports the information coded into DNA out of the nucleus in order to drive the synthesis of proteins in the cytoplasm: DNA → RNA → Protein. The other well known RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA) which are also intimately connected with the protein synthetic machinery. However, over the past two decades, it has become increasingly clear that RNA serves a range of functions, of which protein coding is only one part. Some regulate gene expression, others act as enzymes, some are even crucial in the formation of gametes. These are called non-coding or ncRNA.

Since RNAP is involved in the production of molecules that have such a wide range of roles, one of its main functions is to regulate the number and kind of RNA transcripts formed in response to the cell’s requirements. A number of different proteins, transcription factors and signaling molecules interact with the enzyme, especially the carboxy-terminal end of one subunit, to regulate its activity. It is believed that this regulation was crucial for the development of eukaryotic plants and animals, where genetically identical cells show differential gene expression and specialization in multicellular organisms.

In addition, the optimal functioning of these RNA molecules depends on the fidelity of transcription – the sequence in the DNA template strand must be represented accurately in the RNA. Even a single base change in some regions can lead to a completely non-functional product. Therefore, while the enzyme needs to work quickly and complete the polymerization reaction in a short span of time, it needs robust mechanisms to ensure extremely low error rates. The nucleotide substrate is screened at multiple steps for complementarity to the template DNA strand. When the correct nucleotide is present, it creates an environment conducive to catalysis and the elongation of the RNA strand. Additionally, a proofreading step allows incorrect bases to be excised.

There is remarkable similarity in the RNA polymerases found in prokaryotes, eukaryotes, archea and even some viruses. This points to the possibility that they evolved from a common ancestor. Prokaryotic RNAP is made of four subunits, including a sigma-factor that dissociates from the enzyme complex after transcription initiation. While prokaryotes use the same RNAP to catalyze the polymerization of coding as well as non-coding RNA, eukaryotes have five distinct RNA polymerases.

Eukaryotic RNAP I is a workhorse, producing nearly fifty percent of the RNA transcribed in the cell. It exclusively polymerizes ribosomal RNA, which forms a large component of ribosomes, the molecular machines that synthesize proteins. RNA Polymerase II is extensively studied because it is involved in the transcription of mRNA precursors. It also catalyzes the formation of small nuclear RNAs and micro RNAs. RNAP III transcribes transfer RNA, some ribosomal RNA and a few other small RNAs and is important since many of its targets are necessary for normal functioning of the cell. RNA polymerases IV and V are found exclusively in plants, and together are crucial for the formation of small interfering RNA and heterochromatin in the nucleus.

Basics of DNA Replication

Figure 4. The three suggested models of DNA replication. Grey indicates the original DNA strands, and blue indicates newly synthesized DNA.

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. This model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested: conservative, semi-conservative, and dispersive (see Figure 4).

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands act as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N) that gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 5).

Figure 5. Meselson and Stahl experimented with E. coli grown first in heavy nitrogen ( 15 N) then in 14 N. DNA grown in 15 N (red band) is heavier than DNA grown in 14 N (orange band), and sediments to a lower level in cesium chloride solution in an ultracentrifuge. When DNA grown in 15 N is switched to media containing 14 N, after one round of cell division the DNA sediments halfway between the 15 N and 14 N levels, indicating that it now contains fifty percent 14 N. In subsequent cell divisions, an increasing amount of DNA contains 14 N only. This data supports the semi-conservative replication model. (credit: modification of work by Mariana Ruiz Villareal)

The E. coli culture was then shifted into medium containing 14 N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was centrifuged at high speeds in an ultracentrifuge. Some cells were allowed to grow for one more life cycle in 14 N and spun again. During the density gradient centrifugation, the DNA is loaded into a gradient (typically a salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its density in the gradient. DNA grown in 15 N will band at a higher density position than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the other two modes were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Art Connections

Figure Errors in splicing are implicated in cancers and other human diseases. What kinds of mutations might lead to splicing errors? Think of different possible outcomes if splicing errors occur.

Figure Mutations in the spliceosome recognition sequence at each end of the intron, or in the proteins and RNAs that make up the spliceosome, may impair splicing. Mutations may also add new spliceosome recognition sites. Splicing errors could lead to introns being retained in spliced RNA, exons being excised, or changes in the location of the splice site.

Watch the video: Evidences of RNA as Genetic material (July 2022).


  1. Eaton

    I'm sorry, but in my opinion, you are wrong. I am able to prove it. Write to me in PM, discuss it.

  2. Kemuro

    They are wrong. I am able to prove it.

  3. Donell

    Follow the pulse of the blogosphere on Yandex Blogs? It turns out that Tatiana's day is coming soon.

  4. Baucis

    Thanks, can I also help you with something?

  5. Gogar

    Bravo, it seems to me, is the admirable phrase

  6. Wakil

    You are not right. I'm sure. Write in PM, we will talk.

  7. Mountakaber

    I think, that you are mistaken. I can prove it. Write to me in PM.

Write a message