Meeting Report

Protein translation is the elegant process by which the genetic code is used to produce enzymes, structural proteins, and other working components of the cell. This process is carried out by ribosomes, intracellular organelles composed of RNA and protein that are charged with making polypeptides quickly and accurately, using messenger RNAs (mRNAs) as templates. The process can be divided into a series of phases: binding of an mRNA, initiation of peptide synthesis by binding a transfer RNA (tRNA) at the initiation codon, elongation by the formation of peptide bonds between amino acids as subsequent tRNAs are bound, termination and release when the polypeptide is finished, and ribosome recycling so that the organelle is in the proper conformation to start the next cycle of synthesis. Proteins, in the form of initiation factors, elongation factors, and release factors, facilitate and regulate these phases and the transitions between them.

This complex process has been studied with a wide variety of methods, including biochemical, biophysical, molecular biological, and genetic. X-ray crystallography has yielded finely detailed structures on to which these functions can be mapped. Current knowledge of the basic process is highly detailed, yet there is still much more to find out. On April 1, 2009, three ribosome researchers came together at the New York Academy of Sciences to present their latest findings on the inner workings of this amazing molecular machine.

A process of selection

Marina V. Rodnina and her colleagues at the Max Planck Institute for Biophysical Chemistry are studying the mechanisms by which ribosomes select the mRNA templates that they will translate. The selection of mRNAs offers a chance for the ribosome, and the cell as a whole, to regulate how and when each type of mRNA is translated. Some mRNAs encode proteins that are needed continually, while others encode proteins that are needed only at certain times for specific events in the cell cycle or during differentiation. Some proteins are needed in large quantities, while others are only needed in small amounts.

Rodnina and her group are studying the formation of the initiation complex, a macromolecular complex that in bacteria is made up of the smaller, 30S subunit of the ribosome, an mRNA molecule, an f-methionyl-tRNA that starts translation, and three proteins, known as initiation factors IF1, IF2, and IF3. They are studying the order in which this complex is assembled and the kinetics of each step, to determine which features of the process govern how the ribosome selects an mRNA for translation.

The translation initiation region of each mRNA includes sequences and structures that could affect this process, for example, the sequence of the Shine-Dalgarno site, which is responsible for binding to the ribosome, the spacer between this site and the initiation codon, and any secondary structure that is present in the mRNA molecule.

There are two checkpoints at which mRNA selection is controlled, mostly by kinetic means.

The group has found that there are two checkpoints at which mRNA selection is controlled, predominantly by kinetic means: the formation of the initiation complex, and the formation of the full 70S ribosomal particle after the 50S subunit has been added to this complex. The first checkpoint appears to be governed by the presence of secondary structure in the mRNA. RNAs with no secondary structure are translated efficiently, while those with extensive secondary structure form unproductive initiation complexes that quickly dissociate without proceeding to translation.

Using a series of test mRNAs with different Shine-Dalgarno sequences, spacers, and start codons, they have also identified structures in the mRNA that affect the kinetics of the transition from the 30S to the 70S ribosomal particle. This step is required for elongation to begin. The strength of the Shine-Dalgarno interaction with the ribosome, the length of the spacer, and the fidelity of the codon-anticodon interaction with the initiating tRNA were all found to affect not only the interaction between the ribosome and the mRNA, but also the efficiency of later steps in the elongation process, including the formation of the first peptide bond. These effects were mediated by the initiation factors IF1 and IF3.

An mRNA with a strong Shine-Dalgarno sequence or a noncognate or near-cognate codon will slow down the transition to translation.

Once elongation begins, the ribosome must also select the correct tRNAs in sequence. This step is crucial to the formation of the correct product, and despite rapid polypeptide synthesis, amino acid misincorporation occurs at a very low rate. Here Rodnina and her group studied how the ribosome handles codon-anticodon matches that are one base off (near-cognate) or completely mismatched. They found that tRNAs bind with different stabilities depending on exactness of codon-anticodon match, and that the resulting geometry allows the ribosome to recognize whether the correct tRNA has been selected. Binding of the correct tRNA is stabilized by induced fit, that is, changes in the structure of the ribosome and elongation factors that bind it even more closely once it is in place. These changes accelerate subsequent steps of the elongation process. Binding of a near-cognate, on the other hand, does not lead to induced fit and results in rapid dissociation from the ribosome. Incorrect tRNA selections proceed through the process at much slower rates, if at all, in a kind of "kinetic partitioning" that selects for the desired tRNAs and promotes formation of the correct product.

Mistakes will be made

Despite the existence of these checkpoints, ribosomes do sometimes make mistakes, binding the wrong tRNA and allowing a missense amino acid to be incorporated. Hani Zaher and his colleagues of the Johns Hopkins School of Medicine are studying the process by which such mistakes are kept out of the final peptide. This process is very important because a single missense mutation in an important protein can lead to serious deleterious consequences. Zaher cited the example of the sticky mutation in mouse, a missense mutation in the editing domain of the enzyme alanyl tRNA synthetase, which leads to neurodegeneration in the brain and ataxia, a gross lack of coordination.

Peptides that included mismatches were released from the ribosome, even though no stop codon was present.

Zaher is using an in vitro bacterial translation system to investigate what happens when ribosomes are called upon to make short peptides using tRNAs with cognate, near-cognate, or completely mismatched anticodons. Somewhat unexpectedly, they observed that peptides that included mismatches were released from the ribosome, even though there was no stop codon present in the mRNA. This result was unexpected because normally the release factors that control peptide release after full translation are very adept at recognizing stop codons and allowing peptides to be released only at the proper time. Further experimentation with the system revealed that release factors RF2 and RF3 were involved in promoting the release of defective products. When a misincorporation has occurred, these release factors bind with high affinity and cause premature termination of translation.

Zaher has also detected another mechanism that leads to the rapid release of peptides containing misincorporated amino acids. During elongation, the growing polypeptide chain and its accompanying tRNA is moved from the A, or acceptor site, to the P, or peptidyl site, to allow the tRNA carrying the next amino acid to bind to the acceptor site. They found that the presence of a mismatch between the codon and anticodon at the P site compromised the fidelity of correct selection of the next tRNA at the A site, so that an incorrect tRNA was allowed to bind 5% to 50% of the time. Each cycle in which this occurs increases the likelihood that the defective peptide will be recognized by the release factors and released without further elongation. Zaher suggested that such a system would be particularly useful to the cell during times of amino acid starvation and cellular stress, since it would prevent the cell from using up valuable amino acids in defective proteins.

Machine dynamics

The ribosome is a molecular machine with a number of highly mobile parts that power the assembly line of mRNA translation and protein synthesis. Ruben L. Gonzalez, Jr. and his group at Columbia University are studying how the dynamics of the required conformational changes control the process of protein synthesis. They are using fluorescence resonance energy transfer (FRET), a visualization method that allows them to follow changes in the relationship between two fluorescent tags attached at different places on the ribosome as the parts of the ribosome move. Their experiments are performed on single ribosomes held on a flat surface, which allows them to follow these movements in real time on a millisecond to hours timescale.

tRNAs bound to the A and P sites oscillate in a dynamic equilibrium between two configurations.

Much of their work have been dedicated to the process of translocation, which is the movement of the tRNA holding the nascent polypeptide between the ribosomal A and P sites. They have found that the tRNAs bound to the A and P sites oscillate in a dynamic equilibrium between two configurations, and that these changes in tRNA configuration are coupled to structural rearrangements of the pretranslocation ribosmal complex, specifically conformational changes of a highly mobile ribosomal part known as the L1 stalk. Their experiments indicate that the entire pretranslocation ribosomal complex has two global states, GS1 and GS2, that are defined by these coupled tRNA and L1 stalk dynamics. These dynamics are in turn coupled to the ratcheting mechanism of the ribosome as it moves along the mRNA and polypeptide elongation proceeds.

Together with results from other laboratories, these results have led to new insight into the roles of the elongation factors, which appear to influence this equilibrium and thus influence the dynamics of the process as a whole. Indeed, Gonzalez presented new data using additional translation factors that suggests that translation factors as a whole play a very important role in controlling the global state of the ribosome and thus regulating the overall process of protein synthesis.

Open Questions

Can the secondary structure of mRNAs be modulated to promote their translation when needed?

What are the structural changes that lead to induced fit when a tRNA with an exactly matching anticodon is bound?

Which steps of the tRNA selection are affected by the P-site mismatch?

How is release factor activity modulated?

How is elongation factor activity modulated?

How does the cell use global changes in ribosomal structure to regulate protein translation in specific circumstances?