Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

Abstract

Minigenes encoding the peptide Met-Arg-Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the 'mini-ORFs' employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA(Arg4), CGA is recognized by the abundant tRNA(Arg2). Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNA(Arg4) and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNA(Arg4) but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNA(Arg4) was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome.

Figure 1

Map of the constructs used in this work. Upon addition of the gratuitous inducer IPTG the Lac repressor encoded by lacI^q gene (open arrow) dissociates from the operator region O_lac (grey box) and transcription initiates at promoter P_tac (bold arrow). Transcription presumably terminates at the transcriptional terminator T_rrnb (grey box) The minigene ORFs encode the tripeptide Met–Arg–Arg from the initiator, ATG, through combinations of the triplets AGA and CGA in pairs at codon positions 2 and 3 to the terminator codon TAA. The transcripts are translated from a Shine–Dalgarno sequence (SD, underlined) appropriately spaced from the initiator triplet AUG. The constructs were selected by resistance to ampicillin (Ap^r) conferred by the gene β-lac which encodes β-lactamase. The relative position of the replication origin in the construct (ori) and the position of the restriction sites employed are indicated (see Materials and Methods).

Figure 2

Distribution of tRNA^Arg variants and the effect of puromycin on the peptidyl-tRNA associated with ribosomes. (A) Cell-free extracts from P90C transformants induced for expression of minigenes ATG AGA AGA TAA (AGA AGA) and ATG AGA TAG (AGA TAG) were separated by centrifugation into S100 supernatant (S) and ribosome (R) fractions and further analysed by northern blot (see Materials and Methods). The peptidylated (pep), aminoacylated (aa) and free species of tRNA^Arg4 or tRNA^Arg2 were revealed by blots using ³²P-labelled oligonucleotide probes specific for each tRNA as described in Cruz-Vera et al. (13). (B) The ribosomes obtained from P90C, transformed and induced for minigene expression, were suspended in buffer I with (+ Mg²⁺) or without (− Mg²⁺) magnesium acetate and incubated in the absence (−) or presence (+) of 2.5 mM puromycin for 10 min at 37°C. The tRNA-containing variants were revealed as described above.