The nucleotide in position 32 of the tRNA anticodon loop determines ability of anticodon UCC to discriminate among glycine codons.

Abstract

We have investigated the influence of structures in the tRNA anticodon loop and stem on the ability of the anticodon to discriminate among codons. We had previously shown that anticodon UCC, when placed in the structural context of tRNAG?y from Escherichia coli, discriminated efficiently between the glycine codons, as required by the wobble rules. Thus, this anticodon read GGA and GGG but did not read GGU and GGC, whereas in mycoplasma tRNAGIY, the same anticodon did not discriminate among the glycine codons. We have now determined the reading properties of three constructions based on tRNAlGly containing the anticodon UCC in different structural contexts. In one of these constructs, tRNAlIY-ASL, the anticodon loop and stem are the same as in mycoplasma tRNAGIY. The second construct, tRNAG,'-AS, has an anticodon stem identical with the mycoplasma tRNAGIy, whereas in the last construct, tRNAGly-C32 the only difference from tRNA?GY(UCC) is that the uridine in position 32 of the anticodon loop has been replaced by cytidine. These constructs were tested for ability to read glycine codons in an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each codon. We found that the anticodon UCC, when present in tRNAG'Y-AS, discriminated among the glycine codons, whereas in the constructs tRNAGI1-ASL and tRNA ¶'Y-C32, the same anticodon had lost its ability to discriminate-i.e., it behaved as in mycoplasma tRNAGIY. These results strongly suggest that nt 32 of the anticodon loop of tRNAO'Y(UCC) decisively influences the reading properties of the anticodon UCC.Analysis of tRNA genes and their gene products in mitochondria (1-3) has revealed that in these organelles each family box (group of four codons that have the same nucleotides in the first two positions and that all denote the same amino acid) is read by only one tRNA, which cannot discriminate among the nucleotides in the third-codon position, as required by the wobble rules (4). A similar situation exists in chloroplasts (5, 6), although in this case only about half of the codon families are read without discrimination. Our own analysis of Mycoplasma mycoides indicated that in this microorganism the family boxes, except for the threonine box, are read by only one tRNA each (7-9). The same results have been obtained in Mycoplasma capricolum by Andachi et al. (10). tRNAs that read without discrimination among nucleotides in the third-codon position, in most cases, have an unsubstituted uridine as their wobble nucleotide.These results are reminiscent of a phenomenon we had previously observed in a protein-synthesizing in vitro system programmed with the phage messenger MS2 RNA. In this system we found that family boxes could be read by a single tRNA with an undiscriminating method we referred to as "reading by two-out-of-three" (11). The capacity for this unconventional type of reading varied among different isoaccepting tRNAs. The tRNAGly(UCC) from M. mycoides (12) was almost as efficient in the unconventional reading of cod...

A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift

“…The distribution of the 32-38 pair is biased toward C-A, since it is found in 42%, s 2 C-A in 1%, and U-A in 5% of all sequenced tRNAs. Although both C32-A38 and U32-A38 pairs adopt the same configuration in crystal structures, substitution of a C-A pair by U-A affects the discriminating ability of tRNA transcripts and their frameshifting efficiency (Lustig et al 1993;O'Connor 1998;Auffinger and Westhof 1999). Our results also demonstrate a lowered tRNA Arg mnm 5 UCU stability by an s 2 C32-to-U32 change.…”

Anticodon-dependent conservation of bacterial tRNA gene sequences

“…Subsequent in vivo studies of the translation efficiency of E. coli suppressor tRNAs (Yarus et al 1986;Kleina et al 1990;Yarus and Smith 1995) and in vitro biochemical studies of tRNA binding to the ribosome decoding center (A site) (Lustig et al 1993;Claesson et al 1995;Konevega et al 2004;Olejniczak et al 2005) have chiefly focused on the residues of the anticodon loop. When taken together, the results of these studies show that the residue at position 37, which is adjacent to the third anticodon nucleotide, and the residues of the [32][33][34][35][36][37][38] pseudopair, at the top of the anticodon loop, can affect translation efficiency and binding to the cognate codon.…”

Celebrating wobble decoding: Half a century and still much is new

“…Thus, the unmodified UCC anticodon reads GGA and wobbles to GGG, but does not recognize GGU and GGC. 61

10.1080/15476286.2017.1356562-f0004Figure 4.Sequences of unmodified ASL of tRNA Gly . A. ASLs of E. coli tRNA Gly1 CCC , tRNA Gly2 UCC , and tRNA Gly3 GCC indicating the changes in nucleoside position 32 and 34 (marked in red) from the tRNA Gly1 sequence.

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