Monitoring translation in all reading frames downstream of weak stop codons provides mechanistic insights into the impact of nucleotide and cellular contexts.

A stop codon entering the ribosome A-site is normally decoded by release factors that induce release of the polypeptide. Certain factors influence the efficiency of the termination which is in competition with elongation in either the same (readthrough) or an alternative (frameshifting) reading frame. To gain insight into the competition between these processes, we monitored translation in parallel from all three reading frames downstream of stop codons while changing the nucleotide context of termination sites or altering cellular conditions (polyamine levels). We found that P-site codon identity can have a major impact on the termination efficiency of the OPRL1 stop signal, whereas for the OAZ1 ORF1 stop signal, the P-site codon mainly influences the reading frame of non-terminating ribosomes. Changes to polyamine levels predominantly influence the termination efficiency of the OAZ1 ORF1 stop signal. In contrast, increasing polyamine levels stimulate readthrough of the OPRL1 stop signal by enhancing near-cognate decoding rather than by decreasing termination efficiency. Thus, by monitoring the four competing processes occurring at stop codons we were able to determine which is the most significantly affected upon perturbation. This approach may be useful for the interrogation of other recoding phenomena where alternative decoding processes compete with standard decoding.

Streptomyces rare codon UUA: from features associated with 2 adpA related locations to candidate phage regulatory translational bypassing.

In Streptomyces species, the cell cycle involves a switch from an early and vegetative state to a later phase where secondary products including antibiotics are synthesized, aerial hyphae form and sporulation occurs. AdpA, which has two domains, activates the expression of numerous genes involved in the switch from the vegetative growth phase. The adpA mRNA of many Streptomyces species has a UUA codon in a linker region between 5' sequence encoding one domain and 3' sequence encoding its other and C-terminal domain. UUA codons are exceptionally rare in Streptomyces, and its functional cognate tRNA is not present in a fully modified and acylated form, in the early and vegetative phase of the cell cycle though it is aminoacylated later. Here, we report candidate recoding signals that may influence decoding of the linker region UUA. Additionally, a short ORF 5' of the main ORF has been identified with a GUG at, or near, its 5' end and an in-frame UUA near its 3' end. The latter is commonly 5 nucleotides 5' of the main ORF start. Ribosome profiling data show translation of that 5' region. Ten years ago, UUA-mediated translational bypassing was proposed as a sensor by a Streptomyces phage of its host's cell cycle stage and an effector of its lytic/lysogeny switch. We provide the first experimental evidence supportive of this proposal.

The energy landscape of -1 ribosomal frameshifting.

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3' messenger RNA (mRNA) structures, but how these mRNA structures enhance -1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal -1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

Polysomes Bypass a 50-Nucleotide Coding Gap Less Efficiently Than Monosomes Due to Attenuation of a 5' mRNA Stem-Loop and Enhanced Drop-off.

Efficient translational bypassing of a 50-nt non-coding gap in a phage T4 topoisomerase subunit gene (gp60) requires several recoding signals. Here we investigate the function of the mRNA stem-loop 5' of the take-off codon, as well as the importance of ribosome loading density on the mRNA for efficient bypassing. We show that polysomes are less efficient at mediating bypassing than monosomes, both in vitro and in vivo, due to their preventing formation of a stem-loop 5' of the take-off codon and allowing greater peptidyl-tRNA drop off. A ribosome profiling analysis of phage T4-infected Escherichia coli yielded protected mRNA fragments within the normal size range derived from ribosomes stalled at the take-off codon. However, ribosomes at this position also yielded some 53-nucleotide fragments, 16 longer. These were due to protection of the nucleotides that form the 5' stem-loop. NMR shows that the 5' stem-loop is highly dynamic. The importance of different nucleotides in the 5' stem-loop is revealed by mutagenesis studies. These data highlight the significance of the 5' stem-loop for the 50-nt bypassing and further enhance appreciation of relevance of the extent of ribosome loading for recoding.