Can ribosomes read ssDNA?

Can ribosomes read ssDNA?

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My question is whether translation can be done, either naturally or artificially, through a ribosome reading (single-stranded) DNA directly. If not, I would like to know what allows ssRNA to be translated but not ssDNA.


Messenger RNAs that are recruited to the ribosome for protein synthesis in vivo, need to satisfy particular structural requirements and must interact with the protein initiation factors that deliver them to the ribosome. Generic single-stranded DNA (ssDNA) does not have these structural characteristics and so cannot be translated on ribosomes under natural conditions.

A single reported attempt to translate deoxyribose-based analogues of these mRNAs under conditions equivalent to those within the cell was unsuccessful. Thus, although experiments suggest that ssDNA may be able to bind transfer-RNA and allow some polypeptide synthesis under non-physiological conditions, this reflects only those aspects of protein biosynthesis that involve base-pairing interactions between the message and other RNAs involved in translation. In contrast, the stages involved in the selection of the first initiation codon and the recognition of termination codons involve interactions between protein factors and the message, where recognition of a hydroxyl group in the 2ʹ-position (and discrimination between thymine and uracil) may well occur and would prevent translation of a synthetic ssDNA message.

If, in fact, no discrimination against DNA has evolved for the ribosomal- and transfer-RNA components of the translation apparatus, this may be because the system arose in an 'RNA World' before DNA evolved, and because free ssDNA in the cell has never been a competitor with mRNA for ribosomes, especially after sophisticated protein-based systems arose for the selection of the appropriate initiation codon.

Contemporary physiological interactions of mRNA with ribosomes

Translation of mRNA by ribosomes (protein biosynthesis) is a complex process which can be divided into a number of stages, each generally involving a variety of RNA and protein molecules - initiation factors (IF), elongation factors (EF) and termination or release factors (RF). The reader who is not familiar with this is advised to read a text-book account such as that in Chapter 29 of Berg et al.. However, to summarize briefly, they are: selection of the correct AUG (usually) on the mRNA for initiation, IF-dependent binding of the initiator-tRNA to the P site, EF-dependent and codon-directed binding of an elongator-tRNA, peptide bond formation catalysed by the large ribosomal subunit, EF-dependent translocation, and eventually stop-codon directed and RF-dependent release of the completed polypeptide chain.

The first step, is crucial for the binding of natural mRNAs - the subject of this question. Under natural cellular conditions prokaryotes and eukaryotes have different methods of ensuring that the ribosome interacts with the correct mRNA codon to start translation. In prokaryotes a specific ribosome-binding signal (Shine and Dalgarno sequence) must interact with the 16S rRNA and a particular initiation factor protein is involved in this step. In eukaryotes the main method is recognition of a modified 5ʹ structure on the mRNA and unwinding of secondary structure, also modulated by protein initiation factors.

I am aware of one report of an attempt to translate a DNA-based analogue of such natural mRNA structures under any conditions. This was by Damian et al. (Biochim. Biophys. Res. Commun 385, 296-301 (2009)), and was unsuccessful, even though physical methods indicated binding to the ribosome. I imagine that recognition by the protein initiation factors did not occur but I cannot be sure that base-pairing to the 16S rRNA was also hindered.

Regardless, one can answer the question posed:

The fact that 'random' single-stranded DNA would lack the structural features for selection of the correct initiation codon explains why it would not be translated under natural conditions - it could not bind to the ribosome.

Artificial systems of protein synthesis

The full details of the reactions on the ribosome only emerged gradually. Nevertheless the lack of understanding of the features of natural mRNA required for binding to the ribosome did not prevent either the dissection of subsequent steps of protein biosynthesis, nor the use of ribosomal systems to decipher the genetic code. This is because it was possible to bypass the initial steps by using suitable unphysiological conditions, that allowed polynucleotides or oligonucleotide triplets to bind to the ribosome, where further reactions were possible, albeit generally less efficiently than in the cell. Such conditions included high concentrations of magnesium ions and certain antibiotics that affect the accuracy of protein synthesis. It has been suggested that the neutralization of the charge on the backbone phosphates of the RNA by Mg2+ enhances (non-specific hydrophobic interactions at the mRNA-binding channel, and that the antibiotics stabilize a state of the ribosome in which amino-acyl tRNA can bind more easily (and hence promote the binding of polynucleotides by base pairing). Once bound with the codons hydrogen-bonded to the cognate anticodons of tRNA, subsequent elongation reactions depended on components of the ribosome other than the artificial messenger.

Thus, it was that the genetic code was deciphered using bacterial cell-free systems to which were added simple synthetic polynucleotides, lacking Shine and Dalgarno sequences or start (or stop) codons; and by experiments in which amino acyl-tRNAs were bound to triplet codons in the absence of both IFs and EFs. Other partial reactions of protein synthesis were similarly dissected artificially - the peptidyl transferase reaction was studied on isolated 50S subunits using just a fragment of tRNA and puromycin by conducting the reaction in 50% ethanol!

Experiments with single-stranded DNA

Understanding that ribosomes can be induced to bind and translate 'non-mRNA' oligo-ribonucleotides under certain artificial conditions, we are in a position to consider the significance of reports of translation of oligo-deoxyribonucleotides - ssDNA.

The original experiments in this area by McCarthy and Holland in 1965 showed that denatured DNA from various sources (including animal cells) could stimulate the incorporation of radioactive amino acids in a bacterial cell-free system, but this depended on the presence of antibiotics. Furthermore, it was shown in Khorana's lab that only certain synthetic poly-deoxynucleotides (analogous to the poly-ribonucleotides he used to decipher the genetic code) were active. Thus, it would appear that under the non-physiological conditions that favour promiscuous binding of poly- and oligo-ribonucleotides, there is some binding of ssDNA, and subsequent translation.

A paper by Ricker and Kaji mentioned by @Mesentery - reports that oligodeoxynucleotides could function in some partial reactions (fmet-tRNA binding) although not in others. In particular it was not possible to perform RF-dependent termination reaction with oligonucleotides containing termination codons, but - in the presence of antibiotics - termination independent of release factors did occur.

It is, thus, obvious that these experiments in which ssDNA is translated on ribosomes have no physiological significance. However it is still pertinent to ask the complement to the question of the poster, why does the system work at all?

Why are deoxyribose (and thymine) not discriminated against under artificial conditions?

Enzymes of synthesis and degradation of nucleic acids - RNA and DNA polymerases; ribonucleases and deoxyribonucleases - are specific for either RNA or DNA (or in some cases DNA/RNA hybrids). This is both important to ensure they fulfil their specific functions, and perhaps an inevitable consequence of the nature of their reactions - the making or breaking of sugar-phosphate bonds. Here we are dealing with catalysis by a protein enzyme that binds these structures at its active site.

In contrast, those reactions of protein synthesis for which certain requirements may be relaxed under non-physiological conditions involve base-pairing interactions. In modern ribosomes they may be optimized by associated proteins, but it is thought that the latter only evolved later. Artificial conditions, such as high concentrations of magnesium ions or antibiotics, may be thought of as allowing the essential interactions that existed in 'ur-ribosomes'. And, of course as DNA/RNA hybrids readily form, the participation of DNA in such interactions is not so surprising. (In the RNA world of 'ur-ribosomes' - or even an RNA/protein world - there would be no need to discriminate against DNA, because it had not yet arisen. And chemically, the substitution of a hydrogen for a hydroxyl group could not hinder an interaction - at the worst it would only weaken it.)

It is pertinent that the contemporary AUG-selection and termination reactions depends on RNA-protein interaction. These might well involve recognition of ribose as well as base sequence, explaining the observations by Ricker and Kaji, and by Damian et al., mentioned above.

My question is whether translation can be done, either naturally or artificially, through a ribosome reading (single-stranded) DNA directly.

Ribosomes participate into the translation of mRNA into proteins. See the wikipedia article for more information

If not, I would like to know what allows ssRNA to be translated and ssDNA not.

The "if not" makes no sense because answering yes or no to the first question does not really have much to do with the second question.

ssRNA and ssDNA refers to the genetic material of some viruses. Most of the time, I do not think we use the terms ssDNA and ssRNA to refer to a virus that reverse transcribe the RNA into DNA, so your question is unclear and confusing.

Some viruses do reverse transcribe RNA to DNA. They do so with a reverse transcriptase.

What I want to know with the second question is what structural or chemical characteristic allows the reading of RNA but not DNA.

Oh I think I understand your question…

Double stranded DNA cannot be translated by a ribosome for sure. I suppose ssDNA could eventually fit in a ribosome (or a slightly modified one), however, the tRNA should be adapted to usingTand notU. I am not a molecular biologist and I can't say more than that.

I want to know how DNA lacking a 2-hydroxy group and using thymine instead of uracyl makes it unreadable to a ribosome

I don't know if it does and, if it does, then I don't know why! Sorry, I am not able to help more!

Viral Classification

Since viruses lack ribosomes (and thus rRNA), they cannot be classified within the Three Domain Classification scheme with cellular organisms. Alternatively, Dr. David Baltimore derived a viral classification scheme, one that focuses on the relationship between a viral genome to how it produces its mRNA. The Baltimore Scheme recognizes seven classes of viruses.

In a first for cell biology, scientists observe ribosome assembly in real time

Credit: The Scripps Research Institute

A team of scientists from Scripps Research and Stanford University has recorded in real time a key step in the assembly of ribosomes—the complex and evolutionarily ancient "molecular machines" that make proteins in cells and are essential for all life forms.

The achievement, reported in Cell, reveals in unprecedented detail how strands of ribonucleic acid (RNA), cellular molecules that are inherently sticky and prone to misfold, are "chaperoned" by ribosomal proteins into folding properly and forming one of the main components of ribosomes.

The findings overturn the longstanding belief that ribosomes are assembled in a tightly controlled, step-wise process.

"In contrast to what had been the dominant theory in the field, we revealed a far more chaotic process," says James R. Williamson, Ph.D., a professor in the Department of Integrative Structural & Computational Biology at Scripps Research. "It's not a sleek Detroit assembly line—it's more like a trading pit on Wall Street."

For the study, Williamson's lab collaborated with the lab of Joseph Puglisi, Ph.D., a professor at Stanford University. Although the work is a significant feat of basic cell biology, it should enable important advances in medicine. For example, some current antibiotics work by inhibiting bacterial ribosomes the new research opens up the possibility of designing future antibiotics that target bacterial ribosomes with greater specificity—and thus, fewer side effects.

More generally, the research offers biologists a powerful new approach to the study of RNA molecules, hundreds of thousands of which are active at any given time in a typical cell.

"This shows that we now can examine in detail how RNAs fold while they are being synthesized and proteins are assembling on them," says first author Olivier Duss, Ph.D., a postdoctoral research fellow in the Department of Integrative Structural & Computational Biology at Scripps Research. "This has been a very difficult thing to study in biology because it involves several distinct biological processes that are dependent on each other and have to be detected simultaneously."

The team used an advanced imaging technology called "zero-mode waveguide single-molecule fluorescence microscopy," which they have adapted in recent years for real-time tracking of RNAs and proteins. Ribosomes are made of both RNA and proteins, reflecting a molecular partnership that is widely believed to go back nearly to the dawn of life on Earth.

In a proof-of-principle study published last year, the researchers used their approach to record an early, brief and relatively well-studied stage of ribosome assembly from the bacterium E. coli. This involved the transcription, or copying out from its corresponding gene, of a ribosomal RNA, and initial interactions of this RNA strand with a ribosomal protein.

In the new study, the team extended this approach by tracking not only the transcription of a ribosomal RNA but also its real-time folding. The work provided a detailed look at a complex, and until-now mysterious, part of E. coli ribosome assembly—the formation of an entire major component, or domain, of the E. coli ribosome, with assistance from eight protein partners that end up incorporated into the structure.

A key finding was that the ribosomal protein partners guide the folding of the RNA strand through multiple temporary interactions with the strand, well before they nestle into their final places in the folded RNA-protein molecule. The findings, according to the researchers, also hint at the existence of unknown RNA assembly factors, most likely proteins, that were not present in their lab-dish-type imaging experiments but are present in cells and boost the efficiency of RNA folding.

"Our study indicates that in ribosomal RNA-folding, and perhaps more generally in RNA-folding in cells, many proteins help fold RNA though weak, transient and semi-specific interactions with it," Duss says.

The team will now be able to extend this research further to study not only the rest of ribosome assembly, which involves multiple RNA strands and dozens of proteins, but also the many other types of RNA-folding and RNA-protein interaction in cells.

In principle, this research will yield insights into how RNAs misfold and how such events could be corrected. Scientists believe that many diseases involve or potentially involve the improper folding and related processing of RNAs in cells.

Treatments that already target ribosomes might also be improved. Some current antibiotics, including a class known as aminoglycosides, work by binding to sites on bacterial ribosomes that are not present on human ribosomes. These drugs can have side effects because they also impair the ribosomes of good bacteria, for example in the gut.

"When we understand more fully how bacterial ribosomes assemble and function, we could potentially target them in ways that affect a narrower group of harmful bacterial species and spare the good ones, reducing side effects for patients," Duss says.

Because ribosomes function as protein makers, they are also crucial to the survival of fast-growing tumor cells. Several classes of cancer drug already work by slowing ribosome formation in one way or another. A better understanding of the human ribosome would, in principle, enable its assembly to be targeted more precisely and potently to block cancer growth, Duss notes.

The other co-author of the study, "Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding," was Galina Stepanyuk, PhD, of Scripps Research.

Alert to Biologists: Ribosomes Can Translate the ‘Untranslated Region’ of Messenger RNA

In what appears to be an unexpected challenge to a long-accepted fact of biology, Johns Hopkins researchers say they have found that ribosomes — the molecular machines in all cells that build proteins — can sometimes do so even within the so-called untranslated regions of the ribbons of genetic material known as messenger RNA (mRNA).

“This is an exciting find that generates a whole new set of questions for researchers,” says Rachel Green, Ph.D., a Howard Hughes Medical Institute investigator and professor of molecular biology and genetics at the Johns Hopkins University School of Medicine. Chief among them, she adds, is whether the proteins made in this unusual way have useful or damaging functions and under what conditions, questions that have the potential to further our understanding of cancer cell growth and how cells respond to stress.

In a summary of the findings in yeast cells, was published Aug. 13 in the journal Cell, Green and her team report that the atypical protein-making happens when ribosomes fail to get “recycled” when they reach the “stop” signal in the mRNA. For reasons not yet understood, Green says, “rogue” ribosomes restart without a “start” signal and make small proteins whose functions are unknown.

Ribosomes are made out of specialized RNA molecules (DNA’s chemical cousin) that work together with proteins to read instruction-bearing mRNAs and “translate” their message to create proteins. Each mRNA begins with a “start” code, followed by the blueprint for a specific protein, followed by a “stop” code. And then there’s a segment of code that has always been called the “untranslated region,” because scientists never saw it translated into protein.

But no longer, according to Green and postdoctoral fellow Nicholas Guydosh, Ph.D., who, along with a team at the National Institute of Child Health and Human Development, began the project out of curiosity about a yeast protein called Rli1.

Previous studies had shown that Rli1 can split ribosomes into their two component parts once they encounter a stop code and are no longer needed. This “recycling” process, they say, disengages a ribosome from its current mRNA molecule so that it’s available to translate another one. But it was unclear whether Rli1 behaved the same way in live cells.

To find out, the researchers deprived living yeast cells of Rli1, predicting that translation would slow down as ribosomes piled up at stop codes. To “see” where the ribosomes were, the team added an enzyme to the cells that would chew up any exposed RNA. The RNA bound by ribosomes would be protected and could then be isolated and identified. As predicted, the depletion of Rli1 increased the number of ribosomes sitting on stop codes. But they also saw evidence of ribosomes sitting in the untranslated region, which they called a surprise.

To find out if the ribosomes were actually reading from the untranslated region to create proteins, the team inserted genetic code in that region for a protein whose quantity they could easily measure. Cells with Rli1 didn’t make the protein, but cells missing Rli1 did, proving that their ribosomes were indeed active in the untranslated region.

Further experiments showed that the ribosomes weren’t just continuing translation past the stop code to create an extra-long protein. They first released the regularly coded protein as usual and then began translation again nearby.

“It seems like the ribosomes get tired of waiting to be disassembled and decide to get back to work,” says Guydosh. “The protein-making work that appears right in front of them is in the untranslated region.”

As noted, the purpose of these many small proteins is unknown, but Green says one possibility stems from the fact that ribosomes increase in the untranslated region when yeast are stressed by a lack of food. “It’s possible that these small proteins actually help the yeast respond to starvation, but that’s just a guess,” she says.

Because ribosomes are essential to create new proteins and cell growth, Green notes, scientists believe the rate at which cells replicate is determined, at least in part, by how many ribosomes they have. Cells lacking Rli1 can’t grow because their ribosomes are all occupied at stop codes and in untranslated regions. Thus cancer cells increase their levels of Rli1 in order to grow rapidly.

“We didn’t understand previously how important ribosome recycling is for the proper translation of mRNA,” says Green. “Without it, ribosomes are distracted from their usual work, which is crucial for normal cell maintenance and growth. This finding opens up questions we didn't even know to ask before.”

Other authors of the report include David Young, Fan Zhang and Alan Hinnebusch of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.

This work was supported in part by the National Institute of Child Health and Human Development and the Howard Hughes Medical Institute.


To help reach his goals, Badran has developed what’s known as an “orthogonal translation” system, in which a cell contains both its native ribosomal machinery and a secondary, synthesized ribosome that can only make one protein. The system allows him to tinker with the secondary ribosome without disturbing the original one and killing the cell. This allows him to probe how the ribosomal complex first evolved, what its various components do, and what parts are essential to building proteins.

Badran is also using directed evolution to generate variations of ribosomal parts and transfer RNA molecules, which work with the ribosome to help build proteins. He is using the secondary ribosome to test the capabilities of the new parts he’s building, which he hopes will help the ribosome learn to read quadruplet codons and create novel proteins.

Read more

Badran’s young lab has been busy. They’ve created ribosomes that translate mRNA into proteins more quickly than their natural counterparts, which could help produce biologic medicines more efficiently. They’ve engineered E. coli to host a pool of ribosomes from different microorganisms, which may help scientists develop drugs that target certain pathogens more selectively. They’re also making progress towards a quadruplet-codon system, by deciphering which four-letter codons would correspond to which amino acids.

Badran and his colleagues hope their efforts to reimagine the ribosome will help them learn about the origins of this ancient molecular machine. “I want to create methods that allow us to understand things that exist, and then evolve new tools to study them and perhaps even probe the very limits of biology,” Badran said. “Discovering something that’s been just outside the reach of scientists is what really excites me.”

A New Gene Editing Tool Could Rival CRISPR, and Makes Millions of Edits at Once

With CRISPR’s meteoric rise as a gene editing marvel, it’s easy to forget its lowly origins: it was first discovered as a quirk of the bacterial immune system.

It seems that bacteria have more to offer. This month, a team led by the famed synthetic biologist Dr. George Church at Harvard University hijacked another strange piece of bacteria biology. The result is a powerful tool that can—in theory—simultaneously edit millions of DNA sequences, with a “bar code” to keep track of changes. All without breaking a single delicate DNA strand.

For now, these biological tools, called “Retron Library Recombineering (RLR),” have only been tested in bacterial cells. But as CRISPR’s journey to gene therapy shows, even the weirdest discoveries from lowly creatures may catapult our wildest gene therapy or synthetic biology dreams into reality.

“This work helps establish a road map toward using RLR in other genetic systems, which opens up many exciting possibilities for future genetic research,” said Church.

Wait, Why Is CRISPR Inadequate?

Retrons are weird. Let’s start with CRISPR instead.

You might already be familiar with how it works. There are two components: a type of RNA, and a protein. The “bloodhound” guide RNA tethers the Cas “scissor” protein to a particular gene. In the classic version, Cas chops up the gene to turn it off. More recent advances allow Cas to replace a specific genetic letter, or snip multiple genes at once.

For the chop-and-replace version, as the gene heals itself, it’ll often seek out a template. CRISPR can carry a template gene for the cell to rely on. In this way the cell is tricked into a genetic copy edit: replacing a defective genetic sentence with one that’s biologically grammatical.

The problem with CRISPR is the chopping of DNA. If you’ve ever cut a sentence on your phone, realized you cut the wrong bits, pasted it back with another message that now doesn’t make sense, and hit send—well, that’s kind of analogous to what can happen with CRISPR. The danger of damage to our genome goes up when we need to edit multiple genes. This becomes a massive problem in synthetic biology, which uses genetic manipulation to bestow cells with new abilities, or even engineer completely new organisms.

Cells are stubborn creatures developed from eons of evolution, so changing a single gene is rarely enough to get, for example, a bacteria to pump out biofuels or medications, making multiplexed gene editing necessary. Most cells also rapidly divide, so that it’s essential for any genetic tinkering to stick across generations. CRISPR often struggles with both. The Church team thinks they have a solution.

Meet Retrons

The new tool is called RLR, and the first “R” stands for retrons. These are widespread but utterly mysterious creatures whose “natural biology…is largely unknown,” the team wrote, though similar to CRISPR, they may be involved in the bacterial immune system.

First discovered in 1984, retrons are floating ribbons of DNA in some bacteria cells that can be converted into a specific type of DNA—a single chain of DNA bases dubbed ssDNAs (yup, it’s weird). But that’s fantastic news for gene editing, because our cells’ double-stranded DNA sequences become impressionable single chains when they divide. Perfect timing for a retron bait-and-switch.

Normally, our DNA exists in double helices that are tightly wrapped into 23 bundles, called chromosomes. Each chromosome bundle comes in two copies, and when a cell divides, the copies separate to duplicate themselves. During this time, the two copies sometimes swap genes in a process called recombination. This is when retrons can sneak in, inserting their ssDNA progeny into the dividing cell instead. If they carry new tricks—say, allowing a bacteria cell to become resistant against drugs—and successfully insert themselves, then the cell’s progeny will inherit that trait.

Because of the cell’s natural machinery, retrons can infiltrate a genome without cutting it. And they can do it in millions of dividing cells at the same time.

“We figured that retrons should give us the ability to produce ssDNA within the cells we want to edit rather than trying to force them into the cell from the outside, and without damaging the native DNA, which were both very compelling qualities,” said study author Dr. Daniel Goodman.

The Making of RTR

Similar to CRISPR, RTR has multiple components: the genetic snippet that contains a mutation (the bait), and two proteins, RT and SSAP (reverse transcriptase and single-stranded annealing proteins), that transform the retron into ssDNA and let it insert itself into a dividing cell.

Like Game of Thrones, there’s a lot of players. So to make it clearer: retrons carry the genetic code we want to insert RT makes it into a more compatible form that’s called ssDNA and SSAP sticks it into DNA as it’s dividing. Basically, a Trojan horse invades the cell and pours out spies that insert themselves into the cell—changing its DNA—with the help of enzymatic magicians.

The two proteins are new to the party. Previously, scientists have tried to use retrons for gene editing, but the efficiency was extremely low—around 0.1 percent of all bacterial cells infected. The two newcomers quieted down the bacteria’s natural “alarm system” that corrects DNA changes—so they ignore the new DNA bits—and allow edits to enter and pass on to the next generation. One other trick was to neuter two genes that encode for proteins that normally destroy ssDNA.

In one test, the team found that over 90 percent of bacterial cells readily admitted the new retron sequence into their DNA. They next went big. Compared to CRISPR, retrons have a leg up in that their sequence can act as a bar code. This means it’s possible to perform multiple gene editing experiments at once, and figure out which cells were edited with what retron by sequencing the bar code.

In a proof-of-concept test, the team blasted some bacteria cells with retrons that contained sequences for antibiotic resistance. By sequencing the retron DNA letters alone from a pool of bacteria treated with antibiotics, they found that cells with retrons—giving them the new superpower against drugs—remained in far higher portions than other cells.

In another test, the team tried to determine how many retrons they could use at once. They took another strain of bacteria that’s resistant to antibacterials, and chopped up its genome to build a library of tens of millions of retrons. They then stuck these chunks into hula hoops of DNA—called plasmids—and floated them into bacteria cells. As before, the team could easily find the retrons that conferred anti-bacterial power by sequencing the bar codes of those that remained alive.

But Why?

That’s the how. But what’s the why?

The goal is easy: to find another solution to CRISPR that can influence millions of cells at once, without damaging the cells. In other words, take gene editing into the big data era, through multiple generations.

Compared to CRISPR, the new RLR tool is simpler because it does not require a “guide” tool in addition to an “editing” tool—a retron is basically a two-in-one. Being able to influence multiple genes at once—without physically cutting into them—also makes it an intriguing tool for synthetic biology. The tool’s also got staying power. Instead of a “one and done” CRISPR ethos, it lasts through generations as cells divide.

That said, RTR’s got competition. Because it works best with dividing cells, it might not be as powerful in reluctant cells that refuse to split—for example, neurons. For another, recent upgrades to CRISPR have made it possible to also turn genes on or off—without cutting them—through epigenetics.

But RLR offers scale. “Being able to analyze pooled, barcoded mutant libraries with RLR enables millions of experiments to be performed simultaneously, allowing us to observe the effects of mutations across the genome, as well as how those mutations might interact with each other,” said Church.


Ribosomes are the macromolecular machines that are responsible for mRNA translation into proteins. The eukaryotic ribosome, also called the 80S ribosome, is made up of two subunits – the large 60S subunit (which contains the 25S [in plants] or 28S [in mammals], 5.8S, and 5S rRNA and 46 ribosomal proteins) and a small 40S subunit (which contains the 18S rRNA and 33 ribosomal proteins). [6] The ribosomal proteins are encoded by ribosomal genes.

rRNA found in prokaryotic and eukaryotic ribosomes
Type Size Large subunit (LSU rRNA) Small subunit (SSU rRNA)
prokaryotic 70S 50S (5S : 120 nt, 23S : 2906 nt) 30S (16S : 1542 nt)
eukaryotic 80S 60S (5S : 121 nt, [7] 5.8S : 156 nt, [8] 28S : 5070 nt [9] ) 40S (18S : 1869 nt [10] )

There are 52 genes that encode the ribosomal proteins, and they can be found in 20 operons within prokaryotic DNA. Regulation of ribosome synthesis hinges on the regulation of the rRNA itself.

First, a reduction in aminoacyl-tRNA will cause the prokaryotic cell to respond by lowering transcription and translation. This occurs through a series of steps, beginning with stringent factors binding to ribosomes and catalyzing the reaction:
GTP + ATP --> pppGpp + AMP

The γ-phosphate is then removed and ppGpp will bind to and inhibit RNA polymerase. This binding causes a reduction in rRNA transcription. A reduced amount of rRNA means that ribosomal proteins (r-proteins) will be translated but will not have an rRNA to bind to. Instead, they will negatively feedback and bind to their own mRNA, repressing r-protein synthesis. Note that r-proteins preferentially bind to their complementary rRNA if it is present, rather than mRNA.

The ribosome operons also include the genes for RNA polymerase and elongation factors (used in RNA translation). Regulation of all of these genes at once illustrate the coupling between transcription and translation in prokaryotes.

Ribosomal protein synthesis in eukaryotes is a major metabolic activity. It occurs, like most protein synthesis, in the cytoplasm just outside the nucleus. Individual ribosomal proteins are synthesized and imported into the nucleus through nuclear pores. See nuclear import for more about the movement of the ribosomal proteins into the nucleus.

The DNA is transcribed, at a high speed, in the nucleolus, which contains all 45S rRNA genes. The only exception is the 5S rRNA which is transcribed outside the nucleolus. After transcription, the rRNAs associate with the ribosomal proteins, forming the two types of ribosomal subunits (large and small). These will later assemble in the cytosol to make a functioning ribosome. See nuclear export for more about the movement of the ribosomal subunits out of the nucleus. [11]

Processing Edit

Eukaryotic cells co-transcribe three of the mature rRNA species through a series of steps. The maturation process of the rRNAs and the process of recruiting the r-proteins happen in precursor ribosomal particles, sometimes called pre-ribosomes, and takes place in the nucleolus, nucleoplasm, and cytoplasm. The yeast, S. cerevisiae is the eukaryotic model organism for the study of ribosome biogenesis. Ribosome biogenesis starts in the nucleolus. There, the 18S, 5.8S, and 25S subunits of the rRNA are cotranscribed from ribosomal genes as a polycistronic transcript by RNA polymerase I, [3] and is called 35S pre-RNA. [1]

Transcription of polymerase I starts with a Pol I initiation complex that binds to the rDNA promoter. The formation of this complex requires the help of an upstream activating factor or UAF that associates with TATA-box binding protein and the core factor (CF). Together the two transcription factors allow the RNA pol I complex to bind with the polymerase I initiation factor, Rrn3. As the pol I transcript is produced, approximately 75 small nucleolar ribonucleoparticles (snoRNPs) facilitate the co-transcriptional covalent modifications of >100 rRNA residues. These snoRNPs control 2’-O-ribose methylation of nucleotides and also assist in the creation of pseudouridines. [1] At the 5’ end of rRNA transcripts, small subunit ribosomal proteins (Rps) and non-ribosomal factors assemble with the pre-RNA transcripts to create ball-like knobs. These knobs are the first pre-ribosomal particles in the small (40S) ribosomal subunit pathway. [1] The rRNA transcript is cleaved at the A2 site, and this separates the early 40S pre-ribosome from the remaining pre-rRNA that will combine with large subunit ribosomal proteins (Rpl) and other non-ribosomal factors to create the pre-60S ribosomal particles. [1]

40S subunit Edit

The transcriptional assembly of the 40S subunit precursor, sometimes referred to as the small subunit processome (SSU) or 90S particle happens in a hierarchical fashion – essentially a stepwise incorporation of the UTP-A, UTP-B, and UTP-C subcomplexes. These subcomplexes are made up of over 30 non ribosomal protein factors, the U3 snoRNP particle, a few Rps proteins, and the 35S pre-rRNA. Their exact role, though has not been discovered. [3] The composition of the pre-40S particle changes drastically once cleavage at the U3 snoRNPA dependent sites (sites A0, A1, and A2) are made. This cleavage event creates the 20S pre-rRNA and causes ribosomal factors to dissociate from the pre-40S particle. U3 is displaced from the nascent 40S by the helicase Dhr1. [12] At this point in the ribosome biogenesis process, the 40S pre-ribosome already shows the “head” and “body” structures of the mature 40S subunit. The 40S pre-ribosome is transported out of the nucleolus and into the cytoplasm. The cytoplasmic 40S pre-ribosome now contains ribosomal proteins, the 20s rRNA and a few non-ribosomal factors. The final formation of the 40S subunit “beak” structure occurs after a phosphorylation and dephosphorylation event involving the Enp1-Ltv1-Rps3 complex and the kinase, Hrr25. Cleavage of the 20S pre-rRNA at the D-site creates the mature 18s rRNA. This cleavage event is dependent on several non-ribosomal factors such as Nob1, Rio1, Rio2, Tsr1 and Fap7. [1]

60S subunit Edit

The maturation of the pre-60S subunit into a mature 60S subunit requires many biogenesis factors that associate and disassociate. In addition, some assembly factors associate with the 60S subunit while others only interact with it transiently. As an overall trend, the maturation of the pre-60S subunit is marked a gradual decrease in complexity. The subunit matures as it moves from the nucleolus to the cytoplasm and gradually the number of trans-acting factors are reduced. [3] The maturation of the 60S subunit requires the help of about 80 factors. Eight of these factors are directly involved with the processing of the 27S A3 pre-rRNA, which actually completes the formation of the mature 5’end of the 5.8S rRNA. The A3 factors bind to distant sites on the pre-RNA as well as to each other. Subsequently, they bring areas of rRNA close together and promote the processing of pre-rRNA and the recruitment of ribosomal proteins. Three AAA-type ATPases work to strip the factors from the maturing 60S pre-ribosome. One of the ATPases is a dynein-like Rea1 protein made up of 6 different ATPase domains that form a ring structure. The ring structure is attached to a flexible tail that happens to have a MIDAS (Metal ion-dependentant adhesion site) tip. The Rea1 interacts with the 60S pre-ribosome via its ring while two substrates, Ytm1 and Rsa1, interact with Rea1 through its MIDAS tip. The role of these substrates has not yet been defined. Both though, along with their interactions, are removed in the maturation process of the 60S pre-ribosome. The other two ATPases, Rix7 and Drg1 also function to remove assembly factors from the maturing 60S subunit. Helicases and GTPases are also involved in the removal of assembly factors and the rearrangement of RNA to form the completed 60S subunit. Once in the cytoplasm (see nuclear export), the 60S subunit further undergoes processing in order to be functional. The rest of the large subunit ribosomal particles associate with the 60S unit and the remaining non-ribosomal assembly factors disassociate. The release of the biogenesis factors is mediated mostly by GTPases such as Lsg1 and ATPases such as Drg1. The precise sequence of these events remains unclear. The pathway of 60S cytoplasmic maturation remains incomplete as far as current knowledge is concerned. [3]

In order for the pre-ribosomal units to fully mature, they must be exported to the cytoplasm. To effectively move from the nucleolus to the cytoplasm, the pre-ribosomes interact with export receptors to move through the hydrophobic central channel of the nuclear pore complex. [3] The karyopherin Crm1 is the receptor for both ribosomal subunits and mediates export in a Ran-GTP dependent fashion. It recognizes molecules that have leucine-rich nuclear export signals. The Crm1 is pulled to the large 60S subunit by the help of an adapter protein called Nmd3. The adapter protein for the 40S unit is unknown. In addition to Crm1, other factors play a role in nuclear export of pre-ribosomes. A general mRNA export receptor, called Mex67, as well as a HEAT-repeating-containing protein, Rrp12, facilitate the export of both subunits. These factors are non-essential proteins and help to optimize the export of the pre-ribosomes since they are large molecules. [3]

Because ribosomes are so complex, a certain number of ribosomes are assembled incorrectly and could potentially waste cellular energy and resources when synthesizing non-functional proteins. To prevent this, cells have an active surveillance system to recognize damaged or defective ribosomes and target them for degradation. The surveillance mechanism is in place to detect nonfunctional pre-ribosomes as well as nonfunctional mature ribosomes. In addition, the surveillance system brings the necessary degradation equipment and actually degrades the nonfunctional ribosomes. [1] Pre-ribosomes that build up in the nucleus are destroyed by the exosome, which is a multisubunit complex with exonuclease activity. If defective ribosomal subunits do happen to make it out of the nucleolus and into the cytoplasm, there is a second surveillance system in place there to target malfunctioning ribosomes in the cytoplasm for degradation. Certain mutations in residues of the large ribosome subunit will actually result in RNA decay and thus degradation of the unit. Because the amount of defects that are possible in ribosome assembly are so extensive, it is still unknown as to how the surveillance system detects all defects, but it has been postulated that instead of targeting specific defects, the surveillance system recognizes the consequences of those defects – such as assembly delays. Meaning, if there is a disruption in the assembly or maturation of a mature ribosome, the surveillance system will act as if the subunit is defective. [3]

Mutations in ribosome biogenesis are linked to several human ribosomopathy genetic diseases, including inherited bone marrow failure syndromes, which are characterized by a predisposition to cancer and a reduced number of blood cells. Ribosomal dysregulation may also play a role in muscle wasting. [13]


The ribosome gets started in a process called initiation. Several initiation factor proteins deliver the mRNA to the small subunit, line up the first tRNA, and guide the association with the large subunit. This structure ( 4v4j ), shows a special sequence in the mRNA, called the Shine-Delgarno sequence after its discoverers, which is associated with the last part of the RNA chain in the small subunit. This lines up the mRNA in the right place, making it ready for a special initiator tRNA. In the picture, the little piece of mRNA is shown in red and tRNA is shown in yellow.

A note about the picture: the mRNA, tRNA and protein factors all bind inside the ribosome, between the two subunits, so it is tricky to create a picture that shows what is happening. These pictures show the mRNA, tRNA and other molecules, along with a lightened picture of the ribosome to show their placement in the whole complex.




Exploring the Structure

Ribosome Decoding Center (PDB entry 2wdg)

The new structures of intact 70S ribosomes reveal the secret of life. This illustration shows a closeup of the "decoding center" of the ribosome (PDB entry 2wdg ). This is the place where an incoming tRNA anticodon (shown in yellow) is matched with an mRNA codon (shown in red). As you might imagine, it is essential that this match is perfect, so that only the proper tRNA is paired, and thus that only the correct amino acid is added to the growing chain. The ribosome uses several interactions to test this pairing, ensuring that the base pairing is correct. To look closely at these interactions, click on the image for an interactive Jmol.

Topics for Further Discussion

  1. The ribosome is composed of two subunits that assemble around the mRNA into a functional complex. What are the advantages of this? Can you find other examples of molecules that surround RNA or DNA strands?
  2. Ribosomes are challenging molecules to study. As you are exploring the ribosome
  3. structures in the PDB, compare the different types of data that are used to support the structures, including crystallographic structures at atomic and near-atomic resolution and electron micrograph reconstructions at lower resolution.

Related PDB-101 Resources

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  • Browse Molecular Evolution
  • Browse Central Dogma


  1. A. Korostelev and H. F. Noler (2007) The ribosome in focus: new structures bring new insights. Trends in Biochemical Sciences 32, 434-441.
  2. T. A. Steitz (2008) A structural understanding of the dynamic ribosome machine. Nature Reviews Molecular Cell Biology 9, 242-253.
  3. T. M. Schmeing and V. Ramakrishnan (2009) What recent ribosome structures have revealed about the mechanism of translation. Nature 461, 1234-1242.
  4. E. Zimmerman and A. Yonath (2009) Biological implications of the ribosome's stunning stereochemistry. ChemBioChem 10, 63-72.

January 2010, David Goodsell

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Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.

Keywords: cap-tethering eukaryotic initiation factor mRNA cap reinitiation ribosome footprinting scanning translation initiation translational regulation.

Distinct regulatory ribosomal ubiquitylation events are reversible and hierarchically organized

Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role and fate of ubiquitylated proteins remain unclear. We demonstrate that uS10 and uS5 ubiquitylation are dependent upon eS10 or uS3 ubiquitylation, respectively, suggesting that a hierarchical relationship exists among RRub events establishing a ubiquitin code on ribosomes. We show that stress dependent RRub events diminish after initial stimuli and that demodification by deubiquitylating enzymes contributes to reduced RRub levels during stress recovery. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and can limit RQC activation. Critically, cells lacking USP21 or OTUD3 have altered RQC activity and delayed eS10 deubiquitylation indicating a functional role for deubiquitylating enzymes within the RQC pathway.

Keywords: OTUD3 USP21 ZNF598 cell biology deubiquitylating enzymes human ribosome ubiquitylation ribosome-associated quality control.

Plain Language Summary

Ribosomes are cellular machines that build proteins by latching on and then reading template molecules known as mRNAs. Several ribosomes may be moving along the same piece of mRNA at the same time, each making their own copy of the same protein. Damage to an mRNA or other problems may cause a ribosome to stall, leading to subsequent collisions. A quality control pathway exists to identify stalled ribosomes and fix the ‘traffic jams’. It relies on enzymes that tag halted ribosomes with molecules known as ubiquitin. The cell then removes these ribosomes from the mRNA and destroys the proteins they were making. Afterwards, additional enzymes take off the ubiquitin tags so the cell can recycle the ribosomes. These enzymes are key to signaling the end of the quality control event, yet their identity was still unclear. Garshott et al. used genetic approaches to study traffic jams of ribosomes in mammalian cells. The experiments showed that cells added sets of ubiquitin tags to stalled ribosomes in a specific order. Two enzymes, known as USP21 and OTUD3, could stop this process this allowed ribosomes to carry on reading mRNA. Further work revealed that the ribosomes in cells that produce higher levels of USP21 and OTUD3 were less likely to stall on mRNA. On the other hand, ribosomes in cells lacking USP1 and OTUD3 retained their ubiquitin tags for longer and were more likely to stall. The findings of Garshott et al. reveal that USP21 and OTUD3 are involved in the quality control pathway which fixes ribosome traffic jams. In mice, problems in this pathway have been linked with neurons dying or being damaged because toxic protein products start to accumulate in cells this is similar to what happens in human conditions such as Alzheimer's and Parkinson's diseases. Using ubiquitin to target and potentially fix the pathway could therefore open the door to new therapies.

Watch the video: Your Bodys Molecular Machines (September 2022).


  1. Nootau


  2. Antoine

    Interesting theme, I will take part. Together we can come to a right answer.

  3. Alim

    what we would do without your very good sentence

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