Is release factor 2 uniquely regulated because it is a key transcriptional regulator of other genes?

RF2;Release factor;Frameshift

Smith, Elissa ; Tate, Warren

University of Otago

关键词: RF2;    Release factor;    Frameshift;   

Others  :  https://ourarchive.otago.ac.nz/bitstream/10523/654/1/SmithElissaN2010MSc.pdf

来源: Otago University Research Archive

【 摘 要 】

Prokaryotes contain two release factors, RF1 and RF2, both decode the UAA stop codon but individually they are specific for UAG and UGA respectively. The prfB gene, encoding RF2, uniquely has a sophisticated autoregulation mechanism contained within its mRNA. A frameshift into a +1 reading frame is required to bypass an inframe UGA stop codon located at position 26. In this way the cell is capable of tightly regulating the RF2 concentration, as frameshift is promoted at low cellular levels of RF2. The frameshift mechanism is highly conserved across bacterial species. The prfA gene, encoding RF1, while displaying high levels of sequence homology with prfB and almost complete concordance of function, does not contain a frameshift site. This project set out to test the hypothesis that RF2 has a unique function independent of translation as a regulator of other genes. This was investigated by selecting putative promoter elements from likely candidate genes and cloning them upstream of a β-galactosidase reporter gene. Activity at each promoter was compared under physiologically normal conditions and when release factors were over-expressed. The effect of RF2 over-expression on the Escherichia coli transcriptome as a whole was investigated by microarray analysis. Promoter elements for the three prokaryote release factor genes, prfA, prfB, and prfC (encoding class II release factor RF3) and for the hemA gene (located at the start of the operon containing prfA) were cloned upstream of β-galactosidase. β-galactosidase activity assays in rich medium (LB) showed no activity for the prfA putative promoter. Activity of the prfC promoter appeared to increase with ~5 fold over-expression of RF1. Investigations in M9 minimal medium showed suggestive down-regulation at the hemA and prfB promoter elements when RF2 was over-expressed. Microarray analysis of the E. coli transcriptome investigated gene expression levels in an over-expressing RF2 environment compared to physiologically normal. Changes for the targeted release factor genes studied in the β-galactosidase assays were not significant except for the over-expressed RF2. Surprisingly, the known co-ordinated expression of RF1 and RF2 was not reflected in the microarray results. It was not possible to investigate the effect on the prfB gene, though the over-expression was quantified at <5 fold, a non-physiological level of expression. The majority of genes appeared to decrease in expression as a result of RF2 over-expression. For example, the lysS gene was depressed, suggesting a down-regulation of the prfB-lysS transcript. Other significantly down-regulated genes are involved in acid resistance, transcription, cell division, and ribose transport and metabolism. Significantly up-regulated genes included the leu operon. There is little literature available to connect these processes to RF2. The results of the study did confirm the presence of a previously unidentified promoter upstream of the prfB gene which may be controlling expression of the prfB-lysS transcript. However, there was insufficient evidence to confirm the hypothesis for a second function for RF2 in gene regulation, although interesting suggestive evidence was obtained. Nevertheless the reason for a sophisticated conserved mechanism for regulation of RF2 and not RF1 remains an intriguing mystery.