Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays

Abstract

Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the Ϸ4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although Ϸ80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5 or 3 untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.RNA degradation ͉ RNA half-life ͉ transcript abundance T he rate of mRNA degradation plays a central role in the metabolism of nucleic acids in both prokaryotic and eukaryotic cells (for reviews see refs. 1-5). mRNA decay has been studied in a range of organisms, and much has been learned about the substrate features and ribonucleolytic enzymes that influence mRNA stability. In the case of Escherichia coli, experimenters have demonstrated that extensive variation exists in the rate of decay of individual mRNAs and that specific features of the mRNA sequence as well as transcript secondary structure can be important determinants of such variation (1, 3). Additionally, although the half-lives of some RNAs depend on the physiological state of the cell, as influenced by genetic and environmental factors, the half-lives of other RNAs seem to be independent of cell physiology (6, 7).Much of the information available about RNA decay has been derived from study of RNAs encoded by a very limited number of genes. In fact, we can find published reports of RNA half-life for less than 25 of the 4,288 predicted ORFs in the E. coli genome. Current models of mRNA decay are based on this relatively limited experimental sampling of message turnover. To gain a broader understanding of the fate of mRNAs in E. coli we adapted DNA microarray methodology to measure the decay of each chromosomally encoded mRNA simultaneously.DNA microarrays containing sequences derived ...

Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs

“…However, very few mRNA stability studies have been published. In fact, Bernstein et al (2002) found published reports of RNA half-life for less than 0?5 % of the 4288 predicted ORFs in the E. coli genome. This small number of investigations could reflect the costly and labour-intensive nature of such work.…”

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

“…Cultures of wild-type and hfq − strains were grown at 30 °C to A 600 = 0.4-0.6. Rifampicin (100 µg µl −1 final concentration) was added 56 . Cells were pelleted, resuspended in 200 µl TE (10 mM Tris, pH 8.0, 1 mM EDTA), and passed over QIAshredder mini columns (Qiagen).…”

“…Rifampicin (100 µg µl −1 final concentration) was added and cells were collected at the indicated time points. These samples were added immediately to prechilled tubes containing 0.1 volume phenol stop solution (5% (v/v) buffered phenol, pH 7.4, in ethanol) 56 . Cells were pelleted, resuspended in 200 µl TE (10 mM Tris, pH 8.0, 1 mM EDTA), and passed over QIAshredder mini columns (Qiagen).…”

The compartmentalized vessel

“…In eukaryotes, mRNA localization was suggested to occur by facilitated diffusion in the cytoplasm or by active transport along cytoskeletal filaments. 29,30 The average short half-life of bacterial proteins, 3-8 min, 31 together with the short generation time of bacterial cells suggest that the involvement of active mechanisms for mRNA targeting should be seriously considered. In our study, we measured the half-life of bglF transcripts encoding an integral membrane protein, which turned out to be less than 2 min, 25 reinforcing the need for an active transport mechanism.…”