The structure of an E. coli tRNA_f^Met A₁–U₇₂ variant shows an unusual conformation of the A₁–U₇₂ base pair

Abstract

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A-U base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A-U base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant initiator tRNAA-U, aminoacylated with methionine, in which the C:A mismatch at the end of the tRNA acceptor stem has been changed to an A-U base pair. Sequence alignments show that the mutant tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A-U pair adopts an unusual arrangement. A is in a conformation and forms a single H-bond interaction with U This interaction requires protonation of the N1 atom of A Moreover, the 5' phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G A possible role of this unusual geometry of the A-U pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

Role of aIF1 in Pyrococcus abyssi translation initiation

“…Previous studies have shown that E. coli initiator Met-tRNA f Met A 1 -U 72 variant was a good mimic of Pa-Met-tRNA i Met with identical acceptor stem and anticodon loop ( Supplementary Figure S5 , ( 24 , 30 , 40 )). Therefore, because Met-tRNA f Met A 1 -U 72 tRNA can be obtained with a much higher purity than Pa-Met-tRNA i Met ( 40 ), toeprinting experiments were performed with E. coli initiator Met-tRNA f Met A 1 -U 72 . Control experiments showed very similar toeprint signals with Met-tRNA f Met A 1 -U 72 as compared to Pa-Met-tRNA i Met ( Supplementary Figure S5B ).…”

“…tRNA f Met A 1 -U 72 was produced in PalΔmetZWVΔmetY::kan E. coli strain from PBSTNAV-tRNA f Met A 1 -U 72 , purified and aminoacylated as described ( 39 , 40 ). tRNA i Met , tRNA m Met and tRNA m Met 3G3C were produced in JM101Tr from cloned versions of the tRNAs ( 30 , 41 ), purified and aminoacylated as described ( 39 ).…”

Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation

“…The electron density for the three terminal lysine residues of uS13 is missing. Interestingly, the guanidinium group of uS13-R145 occupies a position corresponding to that of A37 in its "unstacked" conformation observed in the anticodon loop of free initiator tRNA 13,52 . One may imagine that during tRNA accommodation, R145-uS13 selects G30-C40 in the 3 G-C pairs major groove and facilitates the motion of A37 towards the anticodon loop where it is stacked against U36 and A38.…”

“…The strategy used to prepare the IC2 complex was adapted from a previously described one 12 . In brief, archaeal 30S subunits from P. abyssi (Pa-30S) were purified and mixed with IFs Pa-aIF1A, the ternary complex Pa-aIF2:GDPNP:Met-tRNA i Met A 1 -U 72 and a synthetic 26 nucleotide-long mRNA corresponding to the natural start region of the mRNA encoding the elongation factor aEF1A from P. abyssi (A(−17)UUUGGAGGU-GAUUUAAA(+1)UGCCAAAG(+9)) 13 . The complex was purified by affinity chromatography (TALON, Clontech) using N-terminally tagged versions of Pa-aIF2β and Pa-aIF2α.…”

“…Previous biochemical and structural studies of a full IC from Pyrococcus abyssi (30S:mRNA:TC:aIF1:aIF1A) identified two conformations with the initiator tRNA either in a remote position (IC0-P REMOTE ) or bound to the P site (IC1-P IN ). This led us to propose that conformational changes of the TC may participate in start codon selection 12,13 . In our model (named "spring force model" in ref.…”