A Code Within a Code: How Codons Fine-Tune Protein Folding in the Cell.

The genetic code sets the correspondence between the sequence of a given nucleotide triplet in an mRNA molecule, called a codon, and the amino acid that is added to the growing polypeptide chain during protein synthesis. With four bases (A, G, U, and C), there are 64 possible triplet codons: 61 sense codons (encoding amino acids) and 3 nonsense codons (so-called, stop codons that define termination of translation). In most organisms, there are 20 common/standard amino acids used in protein synthesis; thus, the genetic code is redundant with most amino acids (with the exception of Met and Trp) are being encoded by more than one (synonymous) codon. Synonymous codons were initially presumed to have entirely equivalent functions, however, the finding that synonymous codons are not present at equal frequencies in mRNA suggested that the specific codon choice might have functional implications beyond coding for amino acid. Observation of nonequivalent use of codons in mRNAs implied a possibility of the existence of auxiliary information in the genetic code. Indeed, it has been found that genetic code contains several layers of such additional information and that synonymous codons are strategically placed within mRNAs to ensure a particular translation kinetics facilitating and fine-tuning co-translational protein folding in the cell via step-wise/sequential structuring of distinct regions of the polypeptide chain emerging from the ribosome at different points in time. This review summarizes key findings in the field that have identified the role of synonymous codons and their usage in protein folding in the cell.

The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA.

The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free l-Trp-dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of l-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo l-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. In vitro TnaC-peptidyl-tRNA accumulation and toe-printing assays confirmed that in the presence of l-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that l-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce l-Trp-dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of l-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that l-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.

[Synonymous Codon Usage-a Guide for Co-Translational Protein Folding in the Cell].

In the cell, protein folding begins during protein synthesis/translation and thus is a co-translational process. Co-translational protein folding is tightly linked to translation elongation, which is not a uniform process. While there are many reasons for translation non-uniformity, it is generally believed that non-uniform synonymous codon usage is one of the key factors modulating translation elongation rates. Frequent/optimal codons as a rule are translated more rapidly than infrequently used ones and vice versa. Over 30 years ago, it was hypothesized that changes in synonymous codon usage affecting translation elongation rates could impinge on co-translation protein folding and that many synonymous codons are strategically placed within mRNA to ensure a particular translation kinetics facilitating productive step-by-step co-translational folding of proteins. It was suggested that this particular translation kinetics (and, specifically, translation pause sites) may define the window of opportunity for the protein parts to fold locally, particularly at the critical points where folding is far from equilibrium. It was thus hypothesized that synonymous codons may provide a secondary code for protein folding in the cell. Although, mostly accepted now, this hypothesis appeared to be difficult to prove and many convincing results were obtained only relatively recently. Here, I review the progress in the field and explain, why this simple idea appeared to be so challenging to prove.

Transmembrane segments form tertiary hairpins in the folding vestibule of the ribosome.

Folding of membrane proteins begins in the ribosome as the peptide is elongated. During this process, the nascent peptide navigates along 100Å of tunnel from the peptidyltransferase center to the exit port. Proximal to the exit port is a "folding vestibule" that permits the nascent peptide to compact and explore conformational space for potential tertiary folding partners. The latter occurs for cytosolic subdomains but has not yet been shown for transmembrane segments. We now demonstrate, using an accessibility assay and an improved intramolecular crosslinking assay, that the helical transmembrane S3b-S4 hairpin ("paddle") of a voltage-gated potassium (Kv) channel, a critical region of the Kv voltage sensor, forms in the vestibule. S3-S4 hairpin interactions are detected at an early stage of Kv biogenesis. Moreover, this vestibule hairpin is consistent with a closed-state conformation of the Kv channel in the plasma membrane.