Analysis of promoter targets for E. coli transcription elongation factor GreA in vivo and in vitro

Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, NJ; Waksman Institute, Rutgers University, Piscataway, NJ; Department of Biological Sciences, Purdue University, West Lafayette, IN; Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ

^* To whom correspondence should be addressed. Email: serbor{at}aol.com.

	Abstract

Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of E. coli strains lacking Gre factors and strains expressing either wild type (wt) or functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (36-fold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA on transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.