Global RNA Half-Life Analysis inEscherichia coliReveals Positional Patterns of Transcript Degradation

Abstract

Subgenic-resolution oligonucleotide microarrays were used to study global RNA degradation in wild-type Escherichia coli MG1655. RNA chemical half-lives were measured for 1036 open reading frames (ORFs) and for 329 known and predicted operons. The half-life of total mRNA was 6.8 min under the conditions tested. We also observed significant relationships between gene functional assignments and transcript stability. Unexpectedly, transcription of a single operon (tdcABCDEFG) was relatively rifampicin-insensitive and showed significant increases 2.5 min after rifampicin addition. This supports a novel mechanism of transcription for the tdc operon, whose promoter lacks any recognizable binding sites. Probe by probe analysis of all known and predicted operons showed that the 5Ј ends of operons degrade, on average, more quickly than the rest of the transcript, with stability increasing in a 3Ј direction, supporting and further generalizing the current model of a net 5Ј to 3Ј directionality of degradation. Hierarchical clustering analysis of operon degradation patterns revealed that this pattern predominates but is not exclusive. We found a weak but highly significant correlation between the degradation of adjacent operon regions, suggesting that stability is determined by a combination of local and operon-wide stability determinants. The 16 ORF dcw gene cluster, which has a complex promoter structure and a partially characterized degradation pattern, was studied at high resolution, allowing a detailed and integrated description of its abundance and degradation. We discuss the application of subgenic resolution DNA microarray analysis to study global mechanisms of RNA transcription and processing.Gene regulation is a dynamic process which can be controlled by a number of mechanisms as genetic information flows from nucleic acids to proteins. The study of gene regulation in the steady state, while informative, overlooks the underlying dynamics of the processes. Steady-state transcript levels are a result of both RNA synthesis and degradation, and as such, measurements of degradation rates can be used to determine their rates of synthesis (if their steady-state levels are known) as well as reveal regulation which occurs via changes in RNA stability.For the genetic regulatory network of Escherichia coli to be understood and eventually modeled, all means of regulation in use by the cell must be given due attention. RNA degradation in eubacteria was once viewed as a nonspecific, unregulated process. Today it is known to involve multiple degradation pathways, a multisubunit protein complex (the degradosome), and to be an important regulatory mechanism for the expression of some genes (for reviews, see GrunbergManago 1999;Rauhut and Klug 1999;Regnier and Arraiano 2000). A small number of large-scale RNA degradation analyses have recently been reported in budding yeast (Wang et al. 2002), humans (Lam et al. 2001), and E. coli (Bernstein et al. 2002).RNA expression analysis with DNA microarrays has allowed transcription to be st...

In situ transcriptomic analysis of the globally important keystone N2-fixing taxon Crocosphaera watsonii

“…Energy metabolic transcripts may be overrepresented, and putative enzymes and translation transcripts underrepresented in degraded metatranscriptomes (Selinger et al, 2003). Hence, we cannot discount the possibility that the highly expressed isiA and nifH genes are a consequence of mRNA degradation during sample processing.…”

Characterization of the Staphylococcus aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase mRNA Turnover

“…Aliquots were removed at 0, 2.5, 5.0, 15, and 30 min post-transcriptional arrest, and cell viability and rifampin resistance were measured (see Materials and Methods). Total bacterial RNA was isolated, and the mRNA half-lives of transcripts produced in mock-treated, cold-shocked, heat-shocked, stringent response-induced, and SOS response-induced cells were determined using Affymetrix S. aureus GeneChips as previously described (46,49).…”

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil

“…For one, many processes proceed slowly in the environment due to a variety of environmental parameters and limiting factors and therefore require only low amounts of mRNA. In addition, mRNA in active biological matrices is extremely labile, with reported half-lives being as short as just a few minutes (Rauhut and Klug, 1999;Selinger et al, 2003). Further, nucleic acids, including mRNA, are strongly sorbed to soil and sediment components (Chamier et al, 1993), which can significantly affect extraction efficiency.…”