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Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein.

ABSTRACT: High-level expression of non-functional model proteins, derived from elongation factor EF-Tu by the deletion of an essential domain, greatly inhibits the growth of Escherichia coli partly deficient in peptidyl-tRNA hydrolase. High-level expression in wild-type cells has little effect on growth. The inhibitory effect is therefore presumably due to the sequestration of essential tRNA species, partly in the form of free peptidyl-tRNA. The growth inhibitory effect can be modulated by changing the last sense codon in the genes encoding the model proteins. Thus, replacement of Ser by Lys or His at this position increases growth inhibition. The effects of 11 changes studied are related to the rates of accumulation previously observed of the corresponding families of peptidyl-tRNA. Two non-exclusive hypotheses are proposed to account for these observations: first, the last sense codon of mRNA is a preferred site of peptidyl-tRNA drop-off in cells, due to the slow rate of translation termination compared with sense codon translation; secondly, the relatively long pause of the ribosome at the stop codon (of the order of 1 s), results in significant temporary sequestration on the ribosome of the tRNA cognate to the last sense codon.

SUBMITTER: Menez J

PROVIDER: S-EPMC115180 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein.

Menez J J Heurgué-Hamard V V Buckingham R H RH

Nucleic acids research 20001201 23

High-level expression of non-functional model proteins, derived from elongation factor EF-Tu by the deletion of an essential domain, greatly inhibits the growth of Escherichia coli partly deficient in peptidyl-tRNA hydrolase. High-level expression in wild-type cells has little effect on growth. The inhibitory effect is therefore presumably due to the sequestration of essential tRNA species, partly in the form of free peptidyl-tRNA. The growth inhibitory effect can be modulated by changing the la ...[more]

PMID: 11095683

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Project description:Human tRNA(Lys3)(UUU) (htRNA(Lys3)(UUU)) decodes the lysine codons AAA and AAG during translation and also plays a crucial role as the primer for HIV-1 (human immunodeficiency virus type 1) reverse transcription. The posttranscriptional modifications 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)), 2-methylthio-N(6)-threonylcarbamoyladenosine (ms(2)t(6)A(37)), and pseudouridine (?(39)) in the tRNA's anticodon domain are critical for ribosomal binding and HIV-1 reverse transcription. To understand the importance of modified nucleoside contributions, we determined the structure and function of this tRNA's anticodon stem and loop (ASL) domain with these modifications at positions 34, 37, and 39, respectively (hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);?(39)). Ribosome binding assays in vitro revealed that the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);?(39) bound AAA and AAG codons, whereas binding of the unmodified ASL(Lys3)(UUU) was barely detectable. The UV hyperchromicity, the circular dichroism, and the structural analyses indicated that ?(39) enhanced the thermodynamic stability of the ASL through base stacking while ms(2)t(6)A(37) restrained the anticodon to adopt an open loop conformation that is required for ribosomal binding. The NMR-restrained molecular-dynamics-derived solution structure revealed that the modifications provided an open, ordered loop for codon binding. The crystal structures of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);?(39) bound to the 30S ribosomal subunit with each codon in the A site showed that the modified nucleotides mcm(5)s(2)U(34) and ms(2)t(6)A(37) participate in the stability of the anticodon-codon interaction. Importantly, the mcm(5)s(2)U(34)·G(3) wobble base pair is in the Watson-Crick geometry, requiring unusual hydrogen bonding to G in which mcm(5)s(2)U(34) must shift from the keto to the enol form. The results unambiguously demonstrate that modifications pre-structure the anticodon as a key prerequisite for efficient and accurate recognition of cognate and wobble codons.

Dataset Information

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein.

Publications

Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein.

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