Frameshifting as a tool in analysis of transfer RNA modification and translation
Sammanfattning: Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated.During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.
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