Translational accuracy, ribozyme efficiency and NAD metabolism in Escherichia coli
Sammanfattning: Interactions between the translational machinery and mRNA in Escherichia coli have been studied. In the first section nonsense codon readthrough and changed translational reading frame were measured in different growth phases. In early exponential phase, about 7% of –1 frameshift at a U9 slippery sequence is detectable; upon entry into stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% early exponential phase up to 38% at the beginning of stationary phase followed by a decrease. The level of readthrough decreases upon entry into stationary phase. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.The second section is focused on the interaction between a cis-cleaving hammerhead ribozyme (Rz) in an mRNA and a translating ribosome in vivo. It is shown that one of the semi-active constructs can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz sequence in the mRNA.A temperature sensitive mutant, 72c, is analyzed in the third section. The mutant shows a pleoitropic phenotype that indicates disturbances in the transcription or the translation apparatus. The mutation fusB was identified as a frameshift mutation in a nadD gene and renamed nadD72. The gene codes for a nicotinic acid adenylyltransferase (NAMNAT). The analysis of intracellular nucleotide pools showed that very little NAD is produced at the permissive temperature and no synthesis is observed at non-permissive temperature. It was concluded that a small decrease in NAD levels affects ability to grow on minimal medium at 42°C and a large decrease causes a more pleiotropic phenotype. The enzyme was analyzed further; several residues in the active site and at the C-terminus were mutated in an effort to better characterize their roles in catalysis. The data for the C-terminus were not conclusive but amino acid residues His-45, Arg-46 and Thr-11 could be assigned to the active site.
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