I've been reading around Wikipedia recently trying to learn more about various biomechanisms. I'm intrigued by ribosomes - with how small they are, they're basically chemical machines from what I can tell. I've got the general idea of what they do, which is nicely illustrated in the super common GIF below:
My question is how this happens, on a molecular/chemical scale. Even in the GIF, the way the mRNA is being so mechanically fed through the ribosome at the exact-right times seems so deliberate, and I can't imagine a chemical process that does this so well. It seems like we know the exact protein by protein structure of a few types of ribosome; as such, I naively assume we can work out exactly why and how it does each thing it does. I'm sure it's not a simple question - if it's not really answerable in a This Site question, then I guess my question becomes where should I read to learn this.
It is not clear to me that requests for references are on-topic here. In general I would think not, as they are subjective and not about biology per se. I therefore solicit neither votes for, or acceptance of, this answer. I would have used the comment area to provide references, but there isn't really enough space, so I decided to flesh it out into what follows.
Overall perspective of elongation in protein synthesis
The main concern of the question is the movement of mRNA during translation, so I shall focus on elongation (rather than initiation). Most of our detailed knowledge of the ribosome is based on static 'views' of the ribosome at different stages, although there is a paper (reviewed in Nature by Ehrenberg) in which the dynamics have been deduced from millions of time-resolved electron microscopy images. I doubt whether that is what the animation in the Wikipedia article is based, however.
After an introduction to the subject from Wikipedia, I strongly recommend graduating to a good text book. Text books have the advantage over Wikipedia that they are comprehensive, integrated, professionally illustrated and sent out by publishers for review. Berg et al. is probably as detailed as you will find in a general text, the 2002 edition is available online (as are others such as Alberts et al. and Lodish et al.) through NCBI Bookshelf.
Figure 29.24 of Berg et al. provides a static general view of elongation:
You need the associated text to understand this properly, but the step at which the mRNA is thought to move with respect to the ribosome is the penultimate one in the diagram: translocation.
Unfortunately you can't just read through books on NCBI Bookshelf - you have to search for particular topics. But Chapter 29 of Berg et al. is what you need, and the links to the relevant sections are:
How exactly does the mRNA move?
The details of the individual steps in protein synthesis are being deduced from examination of high-resolution structures of complexes at different stages. For the student who as able to go beyond the accounts in general textbooks, a review such as that published in 2009 by Schmeing and Ramakrishnan is perhaps the next port of call. But, chemical virgins beware, this is structural biology, and there is no simple answer that you can scribble on a table napkin!
The ribosome moves: RNA mechanics and translocation
During protein synthesis, mRNA and tRNAs must be moved rapidly through the ribosome while maintaining the translational reading frame. This process is coupled to large- and small-scale conformational rearrangements in the ribosome, mainly in its rRNA. The free energy from peptide-bond formation and GTP hydrolysis is probably used to impose directionality on those movements. We propose that the free energy is coupled to two pawls, namely tRNA and EF-G, which enable two ratchet mechanisms to act separately and sequentially on the two ribosomal subunits.
Two nucleic acids exist in nature, DNA and RNA. Nucleic acids are macromolecules as they are composed of very long chains of repeating subunits, or monomers, called nucleotides. Nucleotides themselves consist of three distinct chemical components: a five-carbon sugar, one to three phosphate groups, and one of four nitrogen-rich (nitrogenous) bases.
In DNA, the sugar component is deoxyribose, whereas in RNA it is ribose. These sugars differ only in that ribose carries a hydroxyl (-OH) group attached to a carbon outside the five-membered ring where deoxyribose carries only a hydrogen atom (-H).
The four possible nitrogenous bases in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). RNA has the first three, but includes uracil (U) in place of thymine. DNA is double-stranded, with the two strands linked at their nitrogenous bases. A always pairs with T, and C always pairs with G. The sugar and phosphate groups create the backbone" of each so-called complementary strand. The resulting formation is a double helix, the shape of which was discovered in the 1950s.
- In DNA and RNA, each nucleotide contains a single phosphate group, but free nucleotides often have two (e.g., ADP, or adenosine diphosphate) or three (e.g., ATP, or adenosine triphosphate).
Ribosome Functions on Endoplasmic Reticulum
What is different about the protein that is destined for the rough endoplasmic reticulum?
[Note: This section describes work that led to a Nobel Prize in Medicine and Physiology to Dr. Gunter Blobel. For more information about Dr. Blobel's work and the pioneering discoveries, click: http://www.nobel.se/medicine/laureates/1999/ ]
The major difference is the fact that it has a hydrophobic signal sequence. This simplified cartoon shows that this is the first part of the protein produced. After the signal sequence is completed, protein synthesis is further inhibited. This is to allow the interaction of the signal sequence with a complex on the rough endoplasmic reticulum. In the above cartoon, note that the signal peptide is allowed to enter and essentially guide the protein into the lumen of the rough endoplasmic reticulum. Once the signal sequence is detected, protein synthesis resumes and the rest of the protein is inserted in the lumen. Note that a signal peptidase near the inner surface of the membrane works to cleave the signal sequence from the growing peptide.
The text reading for this discussion is Alberts et al, Molecular Biology of the Cell, third edition, Garland Publishing, 1994, pp 577-588 (Chapter 12) and pp 599-616.
The complex is actually more complicated than the above. The cartoon to the left shows a view of the signal sequence binding and interaction. Note that the signal sequence is recognized by a Recognition Particle, or SRP. This is then bound to a receptor. This complex guides the protein through a channel like region. It also consists of a docking site for the ribosome.
Another cartoon view of this process shows the signal receptor peptide (SRP) that associates with the large subunit of the ribosome that allows binding to the receptor on the rough endoplasmic reticulum.
After the protein is synthesized, the ribosome dissociates into large and small subunits and the SRP also looses its attachment to the receptor.
Current studies of ribosomal interactions with ER :
Andrea Neuhof, M.M. Rolls, B. Jungnickel, K-U Kalies and T A Rapoport, Binding of Signal Recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction. Molecular Biology of the Cell, 9: 103-115. 1997
- Ø Proteins destined for RER sorting make a signal sequence.
- Ø As signal sequence elongates, it is bound by the signal recognition particle (SRP54) (GTP dependent binding).
- Ø SRP then binds to the SRP receptor (docking protein) on the ER membrane.
- Ø At the same time, ribosomes bind to the RER translocation channel formed by the Sec61p complex.
- Ø This Sec61p complex is a major component of a protein-conducting channel which also includes the “translocating chain-associating membrane protein” or TRAM. This conducts the protein into the RER sac.
Neuhof et al, 1997 have asked the following question: What stimulates the specificity of ribosomal docking?
- Ø Question was asked because ribosomes dock to the Sec61p complex even without the signal sequence. Some clues:
- Ø As the signal sequence begins to appear (short) Ø it can be removed from the ER membrane with high salt concentrations.
- Ø As the signal sequence elongates, Ø ribosome binding is stronger and ribosome complex becomes insensitive to proteases and high salt.
Ø Neuhof's study hypothesized that the presence of the signal peptide was crucial for specific binding. Study Design: ü Added nontranslating ribosomes to compete with translating ribosomes in an in vitro system.
- ü If Signal receptor protein (SRP) was absent, ü the nontranslating ribosomes bound to the Sec61p receptor on ER membranes.
- ü Also, the nontranslating ribosomes competed with the translating ribosomes.
ü Added SRP to bind to the signal peptide.
- ü The translating ribosomes bound tightly and were not displaced.
- ü Then, nontranslating ribosomes failed to compete for the Sec61p receptor sites.
Hence, SRP gives the translating ribosome a competitive edge once it starts translating the signal sequence.
You can see a better surface view in this cartoon. The cartoon is from your text. It shows the Ribosome sitting on the receptor next to the pore. The signal peptide is noted in red. The remainder of the code is read as the ribosome moves along the mRNA. The fluidity of the membrane allows the ribosome to be docked at its receptor site and also move along the mRNA. Each amino acid is added to the growing chain and the polypeptide gets longer.
This cartoon is also from your text. It shows the system without the ribosome. Here you get a better view of the pore through which the protein projects into the lumen and the signal sequence.
How are newly synthesized proteins inserted in the membrane?
Mechanism varies with the type of protein.
Type I: Signal sequence on amino terminus enters first and continues to elongate. Protein is threaded through the translocating channel (open area in rer membrane) until a hydrophobic stop sequence is reached. That hydrophobic stop sequence (seen as a hatched region in the protein) is then inserted in the membrane and forms the anchor for that protein. Signal is cleaved by protease inside the lumen. Type II: No cleavable signal sequence. These proteins have rather long hydrophobic regions that will be anchored in the membrane. Type II proteins are threaded into the lumen with the C terminus leading. Protein continues to be inserted until it reaches the hydrophobic stop signal sequence. Type III: Same as Type II, only the N terminus leads into the lumen.
What regulates the orientation of Type II and III proteins?
Wahlberg, JM. And Spiess, M. Multiple determinants direct the orientation of signal anchor proteins: The topogenic role of the hydrophobic signal domain. J Cell Biology 137: 555-562.
Tested charged amino acids and length of hydrophobic signal sequence. The "positive inside rule" states that amino acid residues nearest the cytosolic side of the hydrophobic anchor sequence are more positive than those nearest the lumenal side. So, whichever end has the least positive charges near the signal anchor patch would go into the ER lumen. One can change the direction of translocation of a protein (reverse it) by mutating the protein and making more positively charged groups near the anchor patch of the other end. Below, the cartoon shows that this can be done to change a Type II protein (COOH end enters ER lumen) to a Type III (which has its amino terminal entering the lumen).
Washburn and Speiss (JCB 137: 555-562, 1997) also tested the length of the hydrophobic signal anchor sequence. The following cartoon shows that a longer hydrophobic anchor sequence (seen as the portion running through the membrane) promotes entry with the amino terminal leading into the lumen.
Proteins destined for insertion into membranes, such as ion channels or receptors have mRNA codes for start and stop sequences that allow multiple passes through the membrane. Signalling sequences (patches) can be formed as described in the above cartoon. It shows the insertion of a double pass transmembrane protein with the loop inside the rough endoplasmic reticulum. The red signal patch has the + charges near the cytosol and starts the insertion process. This continues until the hydrophobic stop signal patch is reached. That anchors the second membrane passage. Note, that both C and N terminal portions are in the cytosol Thus, if this protein is destined for the plasma membrane, that loop in the ER lumen will eventually project outside the cell
Membrane proteins that pass through the membrane multiple times (called "multipass transmembrane proteins) have multiple start and stop signals. They are aligned with the hydrophilic and hydrophobic portions of the lipid bilayer as described in the lecture on membranes. This is shown in the following cartoon
Researchers visualize new states of ribosome translation with cryo-EM
Credit: Pixabay/CC0 Public Domain
The stages in which ribosomes synthesize life-sustaining proteins have been revealed in unprecedented real-time detail by UMass Medical School structural biologists Andrei Korostelev, Ph.D., and Anna Loveland, Ph.D. Their new study of this fundamental molecular mechanism, captured using state-of-the-art, time-resolved, cryo-electron microscopy was published by the journal Nature on July 1.
Ribosomes are the molecular machines that read genetic instructions carried by messenger RNA (mRNA) and translate the instructions into proteins by joining different amino acids. (Amino acids are brought to the ribosomes by corresponding transfer RNAs [tRNAs].)
"Understanding how ribosomes accurately decode mRNA and 'proofread' each tRNA delivered to them was a challenge," said Dr. Korostelev, associate professor of RNA therapeutics. "Time-resolved cryo-EM of protein synthesis on the ribosome showed how proofreading makes translation a very accurate process, which is essential for all life."
The research team, including former Korostelev lab postdoc Gabriel Demo, Ph.D., now a group leader at Masaryk University, used cryo-EM to visualize the delivery of amino-acid-bound tRNA to the ribosome. Visualizing the structural ensembles at different time points provided an unprecedented view of the complete reaction, from initial selection to tRNA proofreading and the addition of an amino acid to the growing protein. Comparison of reactions with correct and incorrect tRNAs uncovered that the small ribosomal subunit strongly holds the correct tRNA and rotates to allow its navigation into the ribosome's catalytic center on the large subunit. By contrast, the small subunit provides almost no support for an incorrect tRNA after initial selection, so it falls off, preventing the addition of an incorrect amino acid to the growing protein.
"I was excited to see so many different structures, or states, including short-lived transient states," said Korostelev. "These transient states are critical because they are the ones in which the decision-making takes place for the ribosome to accept correct tRNAs and reject incorrect ones."
Cryo-EM is a breakthrough technology for visualizing detailed cellular structures, including viruses and ribosomes at near-atomic resolution, with broad applications in structural biology and drug design. Korostelev was one of the faculty members who worked on the proposal for establishing the Massachusetts Cryo-Electron Microscopy Facility at UMMS in 2016. The Korostelev lab, in which Dr. Loveland is a postdoc, studies how cells utilize ribosome complexes to make proteins.
"The big difference between the current work and the past work is that we were able to observe more detailed states that weren't previously visible," said Loveland. "By comparing the correct reaction with the incorrect reaction, we can see how the ribosome favors making correct proteins. In the past, we and others have had snapshots of different parts of the process stalled by inhibitors, whereas now we've visualized a total of 33 states along that pathway and we understand it in a lot more detail."
In the cell, ribosomes can join more than ten amino acids per second, so seeing a complete reaction of amino-acid-bound tRNA selection at near-atomic detail is a challenge. Loveland developed an approach to visualize these reactions with cryo-EM at the UMMS cryo-EM facility.
"We and others previously have had to inhibit—in other words, stall—ribosomal reactions in order to see a single state" she explained. "Now we have found that by slowing down the reaction by cooling it on ice prior to cryo-EM, we're able to resolve so many states that we don't need those inhibitors. This approach allows us to observe what happens over an entire reaction, which is more similar to what actually happens in a cell."
These studies emphasize that high-resolution, time-resolved cryo-EM could become the bona fide structural biochemistry method for visualizing complex biochemical pathways without inhibitors. The ability to visualize reactions with greater accuracy, specificity and detail offers potential for future investigations.(from left) Anna Loveland, PhD, Andrei Korostelev, PhD and GSBS student Christine Carbone examine images in the cryo-EM facility at UMass Medical School. Credit: University of Massachusetts Medical School
"Anna's achievement with time-resolved cryo-EM enabled us to create a 'movie' of the complete mRNA decoding reaction," said Korostelev. "To our knowledge this is the first time so many distinct states have been visualized without an inhibitor. We can now explore other challenging reactions and revisit the studies previously done with inhibitors to learn more."
Among many potential future applications of time-resolved cryo-EM, the technique could be used to see how ribosomes move along the messenger RNA while reading the genetic code. Frame-shifting, in which the ribosomes can slip like gears on viral mRNAs to help viruses make different proteins, is essential for survival and replication of some viruses. These include the now-rampant COVID-19 virus, so its frame-shifting mechanism could be a future drug target.
The 70S Initiation Complex
The initiation phase of protein synthesis involves the formation of a complex between the ribosomal subunits, an mRNA template and tRNAfmet (Fig. 8.5). A 30S subunit attaches to the ribosome-binding site as described above. tRNAfmet then interacts with the AUG initiation codon, and finally the 50S ribosomal subunit attaches. The ribosome is now complete, and the first tRNA and its amino acid are in place in the P site of the ribosome. A 70S initiation complex has been formed, and protein synthesis can begin. The ribosome is orientated so that it will move along the mRNA in the 5' to 3' direction, the direction in which the information encoded in the mRNA molecule is read.
Three proteins called initiation factors 1, 2, and 3, together with the nucleotide guano-sine triphosphate, are needed to help the 70S initiation complex form. Initiation factors 1 and 3 are attached to the 30S subunit. Initiation factor 3 helps in the recognition of the ribosome-binding site on the mRNA. Initiation factor 2 specifically recognizes tRNAfmet and binds it to the ribosome. When the 50S subunit attaches, the three initiation factors are released and the guanosine triphosphate is hydrolyzed, losing its y phosphate to become guanosine diphosphate.
Understanding Ribosomes: Function & Definition
Ribosomes are tiny cellular workshops where proteins, also known as polypeptides, are assembled, in the process known as translation. Messenger RNA, or mRNA, transcribed from DNA, is the template for synthesis of proteins essential for biological function. During translation amino acids are joined together to form a linear polypeptide chain. Some proteins are manufactured on freestanding ribosomes and remain inside the cell. Other proteins are made on ribosomes attached to the surface of the endoplasmic reticulum, a network of membranes and vesicles within the cell. These proteins may be dispatched to the cell membrane or exported for work outside the cell. Ribosomes facilitate protein biosynthesis through recognition of each triple-base codon on the mRNA to be translated. Each codon indicates a specific amino acid to be added to the polypeptide chain. Ribosomes also possess an enzymatic activity responsible for connecting each new amino acid to the growing polypeptide chain. They move along the mRNA in the 5′ to 3′ direction, allowing for the addition of amino acids in a linear fashion.
Ribosomes are protein/RNA complexes made of two subunits. The enzyme peptidyl transferase is located on the larger ribosomal subunit, and catalyzes the formation of a peptide bond, the bond that joins two amino acids. At the beginning of translation the smaller subunit binds to the 5′ end of the mRNA, moving in the 5′ to 3′ direction, scanning the mRNA until it reaches the initiation codon AUG. Here an initiator transfer RNA (tRNA) carrying the amino acid methionone (Met) binds to the initiation codon on the mRNA. Each tRNA contains a single amino acid and an anticodon complementary to the codon of the mRNA. The larger subunit then associates with the smaller subunit for a completed ribosome, and features two binding sites for tRNA, the Aminoacyl (A) site, and the Peptidyl (P) site. Now the Met tRNA is in place, on the P site of the ribosome, ready for another amino acid.
Next a new tRNA molecule containing an amino acid complementary to the next mRNA codon is directed to the A site by proteins known as elongation factors. The anticodon of the tRNA must be complementary to the mRNA codon if chain synthesis is to continue. The ribosome stabilizes the initiating Met tRNA at the P site and the second tRNA at the A site while peptidyl transferase catalyzes formation of a peptide bond. Met then disassociates from the initiating tRNA, leaving one tRNA at the A site with two amino acids attached to it.
The ribosome now moves on to the next mRNA codon. Movement of the ribosome requires an input of energy from ATP. As the ribosome moves, the tRNA carrying the dipeptide shifts from the A site to the P site, and a new tRNA containing an amino acid complementary to the next codon is directed to the empty A site. Peptidyl transferase then catalyzes formation of a second peptide bond, generating a chain of three amino acids. Assembly continues in this manner until the polypeptide is complete. More than one ribosome can participate in polypeptide creation, so that many copies of a protein can be produced from a single mRNA. Stop codons, or nonsense codons UAG, UAA, or UGA, signal the end of protein synthesis. Release factor proteins aid in the disassociation of the ribosome from tRNA, mRNA, and the new polypeptide.
Ribosomes provide a construction site for the building of proteins. Without ribosomal scanning of mRNA, codon-anticodon matching would be a difficult and inefficient process, messenger RNA instructions would go un-translated, and proteins could not be synthesized. Without the catalytic activity of peptidyl transferase, the activation energy required for bond formation would be too great. Proteins perform the work required for survival of the cell, including cell division, metabolism, and cellular respiration. Life as we know it would not be possible without proteins, and proteins would not be possible without that itty-bitty little cellular entity known as the ribosome.
How many ribosomes in a cell?
There are about 10 billion protein molecules in a mammalian cell, and ribosomes produce most of them. A rapidly growing mammalian cell can contain about 10 million ribosomes. This number highly dependent on the cell types and the status of cells.
Ribosomes are particularly abundant in cells that synthesize large amounts of protein. For example, the pancreas is responsible for creating several digestive enzymes, and the cells that produce these enzymes contain many ribosomes. Another example is the immature red blood cells (reticulocytes). Before they become mature, they have to synthesize a lot of hemoglobin. During this differentiation process, ribosomes are particularly abundant in immature reticulocytes.
[In this figure] The reticulocytes contain dark blue dots and curved linear structures (reticulum) in the cytoplasm. They called reticulocytes because the mesh-like network of ribosomal RNA becomes visible under a microscope with certain stains.
Photo credit: Ed Uthman, MD
In the bacterium, a single cell of Escherichia coli (E. coli) contains as many as 15,000 ribosomes and this accounts for one-quarter of the cell’s total mass.
A max-plus model of ribosome dynamics during mRNA translation
Research output : Contribution to journal › Article › peer-review
T1 - A max-plus model of ribosome dynamics during mRNA translation
AU - Brackley, Christopher A.
N1 - A paid open access option is available for this journal. Voluntary deposit by author of pre-print allowed on Institutions open scholarly website and pre-print servers Voluntary deposit by author of authors post-print allowed on institutions open scholarly website including Institutional Repository Deposit due to Funding Body, Institutional and Governmental mandate only allowed where separate agreement between repository and publisher exists Set statement to accompany deposit Published source must be acknowledged Must link to journal home page or articles' DOI Publisher's version/PDF cannot be used Articles in some journals can be made Open Access on payment of additional charge NIH Authors articles will be submitted to PubMed Central after <num>12</num> <period units="month">months</period> Authors who are required to deposit in subject-based repositories may also use Sponsorship Option
N2 - We examine the dynamics of the translation stage of cellular protein production, in which ribosomes move uni-directionally along an mRNA strand, building amino acid chains as they go. We describe the system using a timed event graph—a class of Petri net useful for studying discrete events, which have to satisfy constraints. We use max-plus algebra to describe a deterministic version of the model, where the constraints represent steric effects which prevent more than one ribosome reading a given codon at a given time and delays associated with the availability of the different tRNAs. We calculate the protein production rate and density of ribosomes on the mRNA and find exact agreement between these analytical results and numerical simulations of the deterministic model, even in the case of heterogeneous mRNAs.
AB - We examine the dynamics of the translation stage of cellular protein production, in which ribosomes move uni-directionally along an mRNA strand, building amino acid chains as they go. We describe the system using a timed event graph—a class of Petri net useful for studying discrete events, which have to satisfy constraints. We use max-plus algebra to describe a deterministic version of the model, where the constraints represent steric effects which prevent more than one ribosome reading a given codon at a given time and delays associated with the availability of the different tRNAs. We calculate the protein production rate and density of ribosomes on the mRNA and find exact agreement between these analytical results and numerical simulations of the deterministic model, even in the case of heterogeneous mRNAs.
Ribosome inputs and outputs?
In protein synthesis triplets of RNA are being consumed/evaluated at the ribosome to direct amino acids selection I want to know if the process is destructive to the RNA strand or not or both are possible
Generally no. The ribosome scans the mRNA and coordinates the synthesis of the protein using the triplet code and tRNA-amino acids. After the ribosome passes a region of the mRNA, another ribosome comes along making another copy of that protein from the same mRNA. In this way we can make many copies of proteins from one mRNA.
During translation, an mRNA that has one ribosome on it is called a monosome, while an mRNA that has many ribosomes on it is called a polysome.
That being said, if and when ribosomes are blocked or stalled on the mRNA they can trigger processes called RNA Decay such as Nonsense Mediated RNA Decay, or Staufen Mediated RNA Decay.
The ribosomes are not degrading the mRNA themselves, rather their inability to move forward along the mRNA triggers degradation