Qian Q, Björk G R
Department of Microbiology, Umeâ University, Umeâ, S-90 187, Sweden.
J Mol Biol. 1997 Nov 14;273(5):978-92. doi: 10.1006/jmbi.1997.1363.
A total of 12 Salmonella typhimurium mutants were selected with mutations in the minor tRNAProGGG which suppress a +1 frameshift mutation in the hisD gene. This tRNA normally has 1-methylguanosine (m1G37) next to and 3' of the anticodon (position 37). Since the presence of m1G37 prevents frameshifting, some of the +1 frameshift suppressor derivatives of tRNAProGGG had alterations in the primary sequence abolishing the formation of m1G37. However, several of the mutant tRNAProGGG species had a normal level of m1G37 and a normal-sized anticodon loop, showing that neither m1G37 deficiency, nor an oversized anticodon loop, is a prerequisite for +1 frameshifting. Moreover, base substitutions far from the anticodon, e.g. in the acceptor stem, DHU-loop and stem, and at the top of the anticodon stem, promoted +1 frameshifting. When the frameshifting site (CCC-Uaa; CCC is in the zero frame and a +1 frameshift moves the ribosome to the CC-U codon) is overlapped by a nonsense codon (UAA), the efficiency of frameshifting decreased when release factor 1 was over-expressed and increased at an elevated temperature in a mutant with a temperature-sensitive release factor 1. The frameshifting site (CCC-Uac) was also overlapped with the sense codon UAC, which is decoded by a tRNA species having a 2-methylthio-cis ribozeatin (ms2io6A) at position 37. Mutations in the miaA gene affect the formation of this modified nucleoside and result in an A instead of ms2io6A37 in the tRNA. Such an undermodified tRNA is very inefficient in translation and the efficiency of frameshifting increased in a miaA1 mutant. These results suggest that the frameshifting event occurs at the P-site, since the efficiency of frameshifting was sensitive to the decoding activity of the overlapping codon. We conclude that tRNA with mutations far from the anticodon, with a normal-sized anticodon loop and having m1G37 induce +1 frameshifting at the P-site.
总共筛选出12个鼠伤寒沙门氏菌突变体，它们在次要的tRNAProGGG中发生了突变，该突变可抑制hisD基因中的+1移码突变。这种tRNA在反密码子（第37位）旁边且3'端通常有1-甲基鸟苷（m1G37）。由于m1G37的存在可防止移码，一些tRNAProGGG的+1移码抑制衍生物在一级序列上发生了改变，从而消除了m1G37的形成。然而，几种突变的tRNAProGGG种类具有正常水平的m1G37和正常大小的反密码子环，这表明m1G37缺乏和反密码子环过大都不是+1移码的先决条件。此外，远离反密码子的碱基替换，例如在接受茎、DHU环和茎以及反密码子茎顶部的替换，促进了+1移码。当移码位点（CCC-Uaa；CCC在零框架中，+1移码将核糖体移至CC-U密码子）与无义密码子（UAA）重叠时，当释放因子1过表达时，移码效率降低，而在具有温度敏感型释放因子1的突变体中，在升高的温度下移码效率增加。移码位点（CCC-Uac）也与有义密码子UAC重叠，该密码子由在第37位具有2-甲硫基-顺式核糖玉米素（ms2io6A）的tRNA种类解码。miaA基因中的突变会影响这种修饰核苷的形成，并导致tRNA中A取代ms2io6A37。这种修饰不足的tRNA在翻译中效率非常低，并且在miaA1突变体中移码效率增加。这些结果表明移码事件发生在P位点，因为移码效率对重叠密码子的解码活性敏感。我们得出结论，具有远离反密码子的突变、正常大小的反密码子环且具有m1G37的tRNA在P位点诱导+1移码。
