How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation

Abstract

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

Keywords: mRNA; nascent-peptide chain; protein synthesis; recoding; ribosome; translation control.

Figure 1

Schematic of translation elongation. (Top) For an addition of one amino acid to the growing nascent peptide chain, the conformation and composition of the ribosome cycles through two global states: First, the A-site codon is decoded via (i) initial selection and (ii) proofreading by the incoming aminoacyl-tRNA in complex with EF-Tu and GTP. Proofreading ends with the transfer of the nascent peptide to the amino acid on the accommodated tRNA, which is followed by global conformational changes and translocation (with the help of EF-G complexed with GTP), displaying the next codon in the A site.

Figure 2

Different methods used to study translation. The current model of translation elongation has advanced through combining results from different methods to construct a coherent model. Structural methods such as NMR, x-ray crystallography, and cryoEM provided the structural context of elongation mechanism at the atomic level. Bulk kinetics methods provide the dynamics links between different biochemical and structural states. Single-molecule fluorescence methods bridge structure and dynamics further by observing both simultaneously. Structures shown here were adapted from (16) (PDB entry 5UYK, 5UYL and 5UYM). Bulk kinetics and single-molecule data were adapted from Choi et al (in press).

Figure 3

Example of chemical modifications in mRNA. Among hundreds of known chemical modifications of RNA bases, several modifications occur within the coding region of mRNA can be classified into three categories. First, modifications occurring in the Watson-crick edge of RNA bases that disrupt base-pairing of mRNA (m⁶A and m¹A), and hinder secondary structure formation or codon-anticodon interaction. Second, modifications occurring outside the Watson-Crick edges of the base (Ψ), which do not alter base-pairing ability of the modified base, but could distort the base-pairing of nearby RNA bases. Third, modifications on the ribose of RNA disrupt its interaction with rRNA during decoding (2′-O-methylation).

Figure 4

Interactions within the nascent-peptide exit tunnel (NPET) modulate translation dynamics. Various nascent-peptide elements are known to stall translation of the upstream open-reading frame (ORF) at a precise location to induce rearrangements of mRNA folding and expose the previously sequestered translation start-site of the downstream ORF. The stall can be induced by nascent-peptide elements only, some of which have been shown to slow-down elongation gradually by building multiple contacts within the NPET as the nascent peptide is extended. In addition, interactions with a small molecule can cause an abrupt stall of translation at one codon. Single-molecule data and graphics of NPET were adapted from (148) and (153).

Figure 5

Mechanisms of recoding events involving disrupted translocation and decoding. A. −1 Frameshifting is likely to occur during translocation, impeded by mRNA structure. Translocation into the structured region of mRNA leads to multiple futile bindings of EF-G (shown from smFRET experiments), which may hydrolyze GTP to translocate the codon-anticodon and unfold the mRNA structure simultaneously. The EF-G-bound state may be susceptible to the frameshifting prior to translocation. B. +1 Frameshifting may occur during inefficient decoding, where the A-site substrate in the +1 frame (aa-tRNA ternary complex) may compete with the A-site substrate in the original frame (release factors or another aa-tRNA ternary complex). Intermediate structures and dynamics of +1 frameshifting have yet to be revealed. C. The first stage of bypassing involves impeded decoding by the folding of the nascent-peptide chain within the exit tunnel, which allows mRNA to fold into the vacant A-site and weaken codon-anticodon interaction in the P site, facilitating the take-off of the ribosome from the current P-site codon. Shown structures were adapted from (180). D. After take-off, landing of the ribosome during bypassing may involve several mRNA structures that limit the translocation of the taken-off ribosome. Repeated EF-G binding during landing has been observed in smFRET experiments. Yet the role of EF-G during landing is still unclear.