The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop

Abstract

We have solved to 3.3 Å resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon.

Structural Features of tRNALys Favored by Anticodon Nuclease as Inferred from Reactivities of Anticodon Stem and Loop Substrate Analogs

“…The importance of this configuration is suggested by the weaker reactivity of the IC-rich version of the tRNA (Tables I and II). These results confirm the opposing contributions of the bacterial mnm 5 U34 and mammalian mcm 5 U34 side chains to ACNase reactivity, whereas both modifications similarly influence the overall ASL conformation (12,24). Therefore, the inhibitory effect of mcm 5 U34 on ACNase reactivity suggests a specific interaction between residue Asp 287 of PrrC and the mnm 5 U34 side chain of the natural substrate (11).…”

Primer Unblocking and Rescue of DNA Synthesis by Azidothymidine (AZT)-resistant HIV-1 Reverse Transcriptase

“…Template, Primers, and RT-Natural tRNA 3 Lys was purified from beef liver as described (33). tRNA 3 Lys and ODN, an 18-mer oligodeoxyribonucleotide complementary to the PBS, were radioactively labeled at their 5Ј-ends as described (28).…”

New insights into the formation of HIV-1 reverse transcription initiation complex

“…The D loop contains three guanine nucleotides (G15, G18 and G19) whereas the T loop contains only one ( Figure 3). G15 makes a base pair with C48 and is rather buried within the 3D structure of tRNA 3 Lys [17]. G19 makes a rather weak base-pair with C56, as two alternate conformations are observed in the crystal structure and, as a consequence of this mobility, could be readily accessible to interactions with NC.…”

“…It remains to be established whether the same viral RNA molecule can simultaneously invade the tRNA acceptor stem from both ends or whether some initiate at center of the tRNA while others "zip-in" from the 3'-end, in a statistical manner. Interestingly, within the tRNA 3 Lys tertiary structure [17], C72 and C61 are distant from each other by eleven nucleotides, i.e. exactly one A-helix turn apart and are thus located on the same side of the tRNA acceptor-TΨC helix, i.e.…”

“…G18 makes a hydrogen bond with Ψ55 (O6) and interestingly, the presence of NC in the annealing process is required to induce disappearance of the Ψ55 (N3H) signal. Moreover, bases 16,17 and 20 that surround G18 and G19 are completely exposed to the solvent that render nucleotide in the D-loop easily accessible. Therefore, within the D loop, G18 and G19 appear as likely candidates as being part of the specific NC binding site.…”