Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

Abstract

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb 3+ ions and the selective 2 ′ -hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb 3+ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

“…Our present experiments and recent in-vitro translation experiments performed in S30 extracts 10 showed that essentially no byp was observed with mRNAs that were truncated shortly after the coding gap. These findings appear to contradict previous results obtained using different reporters in vivo, which suggested that translational bypassing was observed even when the sequence beyond the fifth nucleotide after the landing codon was replaced by the reporter sequence 4 .…”

“…Previous work indicated that replacing the gene 60 sequence following the landing site by reporter constructs did not abolish bypassing 4,22 . On the other hand, the bypassing efficiency on truncated gene 60 mRNA in cell lysates was strongly diminished 10 . To assess the minimum length required for bypassing, we tested several mRNA constructs that were truncated at different positions downstream of the landing codon (Fig.…”

“…Next, we asked whether the mRNA region following nt 90 (after codon 30) but preceding the SL element in the 5 0 part of the gap region (nt 91-138) affects bypassing. Prediction of the mRNA structure using mfold and the results of chemical probing 10 suggested the existence of a SL structure upstream of the gap ( Fig. 3a; Supplementary Fig.…”

“…The shortest construct that was efficient in bypassing contained 69 nt following the landing site (Fig. 5b) and included the whole length of a potential secondary structure element (3 0 SL) predicted by computational analysis (mfold) and chemical probing 10 . Translation of the T0 control mRNA, which had the same overall length as the WT construct but contained a stop codon at the indicated position (Fig.…”

“…The take-off codon is 5 0 adjacent to UAG and both are near the top of a stem loop (SL), the 'take-off SL' that is critical for bypassing 4,[7][8][9][10] . The analysis of the mRNA structure suggested that the gap region is structurally autonomous from the upstream and downstream coding regions 10 , consistent with the evolutionary origin of the gap from a mobile element 6 . The presence of the take-off GGA and the matching landing codons, the UAG stop codon next to the take-off GGA and the SL structure in the 5 0 gap are essential for bypassing.…”