One protein from two open reading frames: mechanism of a 50 nt translational bypass

Abstract

Translating ribosomes bypass a 50 nt coding gap in order to fuse the information found in the two open reading frames (ORFs) of bacteriophage T4 gene 60. This study investigates the underlying mechanism by focusing on the competition between initiation of bypassing and termination at the end of the first ORF. While nearly all ribosomes initiate bypassing, no more than 50% resume translation in the second ORF. Two previously described cis-acting stimulatory signals are critical for favoring initiation of bypassing over termination. Genetic analysis of these signals supports a working model in which the first (a stem-loop structure at the junction between the first ORF and the coding gap) interferes with decoding in the A-site, and the second (a stretch of amino acids in the nascent peptide encoded by the first ORF) destabilizes peptidyl-tRNA-mRNA pairing.

Fig. 1. T4 gene 60 bypassing requires matching GGA codons flanking an optimally sized 50 nt coding gap, a stop codon, a stem–loop structure and a nascent peptide signal. In the current model, peptidyl-tRNA₂^Gly detaches from the take-off site GGA, scans the mRNA as it slides through the P-site, and re-pairs with the landing-site GGA.

Fig. 2. The competition between bypassing and termination. (A) Pulse–chase analysis of isogenic strains expressing a GST–gene 60 fusion (pGS1): WT, black bars; a strain expressing a ts RF1, white bars; a strain overexpressing RF1 from a ts promoter, striped bars. The GST–gene 60 fusion and products due to productive (45.1 kDa) or unproductive bypassing (31.7 kDa) are represented schematically to the left of the autoradiograph. The control (C) is an isogenic strain expressing just GST (26.3 kDa). (B) β-galactosidase assay measurements of +6 bypassing (UAG hopping; see bypassing window to the right) demonstrating inactivation of the ts RF1 with increasing temperature.

Fig. 3. Analysis of importance of stop codon context for bypassing efficiency. (A) Gene 60 coding gap and flanking sequence showing mutations (in blue). (B) Pulse–chase analysis of isogenic strains, which differ as indicated, expressing mutant GST–gene 60 constructs. pGS1 is the WT. C (far right) is a strain over-expressing RF1 in which the GST–gene 60 fusion has not been induced. Read-through levels in the strain expressing an amber suppressor tRNA are indicated by blue bars and correspond to the level of single read-through product (see the text). The read-through product for pGS16 is detectable upon more extensive electrophoresis.

Fig. 4. Stimulation of take-off by the stem–loop. (A) Stem–loop and coding gap mutations (in blue). (B) Pulse–chase analysis of isogenic strains, which differ as indicated, expressing mutant GST–gene 60 constructs. Read-through levels in the strain expressing an amber suppressor tRNA (blue bars). Read-through product for pGS16 migrates more quickly than other read-through products, but is visible upon more extensive electrophoresis (not shown).

Fig. 5. Stimulation of take-off by the nascent peptide signal. (A) The amino acid sequences of the nascent peptide signal and nascent peptide signal mutants (blue) were placed in combination with a WT stem or a stem carrying a mutation in the tetraloop. (B) Pulse–chase analysis of isogenic strains, which differ as indicated, expressing mutant GST–gene 60 constructs. Read-through levels in the presence of amber suppressor tRNA are indicated by blue bars. Multiple read-through products are visible for nascent peptide mutants expressed in the amber suppressor strain. These read-through products are also present when the nascent peptide is WT (visible on more extensive electrophoretic analysis), but more apparent in the mutants because the mutation increases the mobility of all GST–gene 60 products.

Fig. 6. (A) Bypassing and UAG hopping (see Figure 2) when RF1 and L9 are inactivated. WT strain, black columns; L9 deficient strain, white columns. (B) Overexpression of RF1 in WT and L9 deficient strains. WT, black striped bars; L9 deficient strain, white striped bars. (C) Bypassing in tRNA₂^Gly mutants when RF1 is mutant or overexpressed. Secondary structure prediction of tRNA₂^Gly shows positions of mutations that reduce bypassing efficiency. Autoradiograph shows pulse–chase analysis of tRNA₂^Gly mutant strains expressing WT gene 60 (pGS1) under conditions in which RF1 is inactive or overexpressed. (D) Influence of ribosomal protein L9 and tRNA₂^Gly on bypassing efficiency with mutant gene 60 variants. WT gene 60 (pGS1; gray bars), a stem mutant (pGS7; red bars), a nascent peptide mutant (pGS12; green bars) and a mutant with an insertion in the coding gap (pGS18; blue bars).