In vitro processing of E. coli tRNA precursors

Using E. coli tRNA precursors isolated from an RNAase P mutant strain, we have studied the steps required for the formation of tRNAs having a mature primary sequence in vitro. Our results suggest that at least three different enzymatic activities can participate in the processing of tRNA precursors.     

Differential repair of premutational UV-lesions at tRNA genes in E. coli.

Summary Ultraviolet radiation produces bacterial revertants that frequently are the result of suppressor mutation. When irradiated cells are incubated under conditions unfavorable for protein synthesis there may be a large decrease in the frequency of observed mutants (mutation frequency decline, or MFD). MFD occurs only in excision-proficient strains and is inhibited by inhibitors of pyrimidine dimer excision. It has therefore been interpreted as enhanced excision of some premutational lesions. Potential de novo UAG suppressor mutation is very susceptible to MFD. Potential conversion mutation, the conversion of a UAG to a UAA suppressor, is at least ten times less susceptible to MFD. A base pair transition at a GC target in a particular tRNA gene is suggested for both de novo suppressor mutation and for conversion mutation. We interpret these results as indicating differential repair of premutational UV photoproducts at two closely spaced sites in the same tRNA gene. The significant difference between these two types of mutation may be the orientation of this target base pair in double helical DNA. The C would be in the transcribed strand of DNA when a nucleic acid alteration produces de novo suppressor mutation. The C would be in the nontranscribed strand, two base pairs removed, when a mutagenic alteration produces suppressor conversion. A model involving facilitated incision by hybridization of the transcribed strand of DNA to its cognate tRNA, under conditions promoting MFD, is described to explain this differential repair.     

Some rRNA operons in E. coli have tRNA genes at their distal ends.

We have previously isolated seven rRNA operons on plasmids or lambda transducing phages and identified various tRNAs encoded by these operons. Each of the seven operons has one of two different spacer tRNA gene arrangements between the genes for 16S and 23S rRNA: either tRNAGlu2 or both tRNAIle1 and tRNAAla1B genes. In addition, various tRNA genes are located at or near the distal ends of rRNA operons. In particular, genes for tRNATrp and tRNAAsp1 are located at the distal end of rrnC at 83 min on the E. coli chromosome. Experiments with various hybrid plasmids, some of which lack the rRNA promoter, have now demonstrated that this promoter is necessary for expression of the distal tRNA genes. Rifampicin run-out experiments have also provided evidence that the tRNATrp gene is located farther from its promoter than the spacer tRNA gene or the 5S RNA gene. These results confirm the localization of genes for tRNATrp and tRNAAsp1 at the distal end of rrnC and strongly suggest that they are co-transcribed with the genes for 16S, tRNAGlu2, 23S and 5S RNA. Other such distal tRNAs have been identified, and it is suggested that they too are part of rRNA operons.