Chromatographic analysis of the aminoacyl-trnas which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV

Abstract

An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the "shifty" tRNAs proposed by Jacks et al. (Cell 55, 447-458, 1988) to carry out frameshifting may be hypomodified isoacceptors.

An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

“…Z. Tsuchihashi and P. Brown (personal communication) have obtained direct evidence for the importance of third-position mispairing for the shiftiness of tRNAs reading the shift site ofEscherichia coli dnaX: introduction ofa tRNA that forms three rather than two base pairs with the final AAG codon in the shift site down regulates frameshifting. Such results suggest that efficient frameshifting on the MMTV shift site depends on an unstable interaction between AAC and the anticodon of tRNAASn, with its modified queosine base (32), since changes of C to U, A, or G reduce frameshifting. The inefficiency of frameshifting on A AAA AAG in a mammalian translation system contrasts dramatically with the remarkably efficient shifting on that sequence in E. coli (17)(18)(19)(20)40), reflecting differences in tRNA species that have doubtless guided the evolution of shift sites.…”

“…These mutations have the effect of changing an AAC (asparagine) codon to another asparagine codon, AAU, or to either of two lysine codons, AAA and AAG. Mammalian cells contain cognate tRNAs for each of the lysine codons but only a single tRNAASn species, most of which has a modified base (queosine) in place of G at the wobble position (32,33). All three changes cause major reductions in frameshift efficiency, from 20% to 4% for AAU and to 2% for the lysine codons.…”

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

“…The presence of Q in the anticodons of these tRNAs (including mitochondrial tRNAs) is compatible with a fundamental role in translation, specifically in modulating fidelity. For example, the À1 frameshifting events essential for correct translation of the retroviral Gag-Pol-Pro polypeptides of human T-cell lymphotropic virus and bovine leukemia virus appear to be dependent on (Q À )-tRNA Asn (Jacks et al, 1988;Hatfield et al, 1989;Carlson et al, 1999Carlson et al, , 2000. Disruption of Q biosynthesis in the pathogenic bacterium Shigella flexneri results in loss of pathogenicity, making several enzymes in its biosynthesis pathway attractive targets for anti-shigellosis drugs.…”

tRNA Modification by S-Adenosylmethionine:tRNA Ribosyltransferase-Isomerase

“…A definitive picture of the biochemical function or functions of queuosine has yet to emerge, but it has been correlated with eukaryotic cell development and proliferation (7)(8)(9)(10), neoplastic transformation (8,(11)(12)(13), tyrosine biosynthesis in animals (14), translational frameshifts essential to retroviral protein biosynthesis (15)(16)(17), and the ability of pathogenic bacteria to invade and proliferate in human tissue (18). Underlying most, if not all, of these phenomena is a role in modulating translational fidelity, consistent with the location of queuosine in the anticodon.…”