Biomedical subjects

Jason A Opdyke

Publications and source records attributed to Jason A Opdyke.

5 recordsLinked to original sources

PubMed2006

Mutational analysis to define an activating region on the redox-sensitive transcriptional regulator OxyR.

The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.

Amino Acid Sequence↗

PubMed2006

Target prediction for small, noncoding RNAs in bacteria.

Many small, noncoding RNAs in bacteria act as post-transcriptional regulators by basepairing with target mRNAs. While the number of characterized small RNAs (sRNAs) has steadily increased, only a limited number of the corresponding mRNA targets have been identified. Here we present a program, TargetRNA, that predicts the targets of these bacterial RNA regulators. The program was evaluated by assessing whether previously known targets could be identified. The program was then used to predict targets for the Escherichia coli RNAs RyhB, OmrA, OmrB and OxyS, and the predictions were compared with changes in whole genome expression patterns observed upon expression of the sRNAs. Our results show that TargetRNA is a useful tool for finding mRNA targets of sRNAs, although its rate of success varies between sRNAs.

Base Pairing↗

PubMed2004

Controlling mRNA stability and translation with small, noncoding RNAs.

Recent studies have led to the identification of more than 50 small regulatory RNAs in Escherichia coli. Only a subset of these RNAs has been characterized. However, it is clear that many of the RNAs, such as the MicF, OxyS, DsrA, Spot42 and RyhB RNAs, act by basepairing to activate or repress translation or to destabilize mRNAs. Basepairing between these regulatory RNAs and their target mRNAs requires the Sm-like Hfq protein which most likely functions as an RNA chaperone to increase RNA unfolding or local target RNA concentration. Here we summarize the physiological roles of the basepairing RNAs, examine their prevalence in bacteria and discuss unresolved questions regarding their mechanisms of action.

Escherichia coli↗

PubMed2004

GadY, a small-RNA regulator of acid response genes in Escherichia coli.

A previous bioinformatics-based search for small RNAs in Escherichia coli identified a novel RNA named IS183. The gene encoding this small RNA is located between and on the opposite strand of genes encoding two transcriptional regulators of the acid response, gadX (yhiX) and gadW (yhiW). Given that IS183 is encoded in the gad gene cluster and because of its role in regulating acid response genes reported here, this RNA has been renamed GadY. We show that GadY exists in three forms, a long form consisting of 105 nucleotides and two processed forms, consisting of 90 and 59 nucleotides. The expression of this small RNA is highly induced during stationary phase in a manner that is dependent on the alternative sigma factor sigmaS. Overexpression of the three GadY RNA forms resulted in increased levels of the mRNA encoding the GadX transcriptional activator, which in turn caused increased levels of the GadA and GadB glutamate decarboxylases. A promoter mutation which abolished gadY expression resulted in a reduction in the amount of gadX mRNA during stationary phase. The gadY gene was shown to overlap the 3' end of the gadX gene, and this overlap region was found to be necessary for the GadY-dependent accumulation of gadX mRNA. We suggest that during stationary phase, GadY forms base pairs with the 3'-untranslated region of the gadX mRNA and confers increased stability, allowing for gadX mRNA accumulation and the increased expression of downstream acid resistance genes.

AraC Transcription Factor↗

PubMed2003

Expression of the secondary sigma factor sigmaX in Streptococcus pyogenes is restricted at two levels.

Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence. A protein (sigma(X)) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor. We report here the isolation of a mutant in which both sigX genes are inactivated, show that sigma(X) functions in GAS cells, and show that the amount of sigma(X) is controlled at two levels. Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a sigma(X)-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells. To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter. Expression of sigX from this promoter led to transcription of the sigma(X)-dependent cinA promoter in GAS cells. We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of sigma(X) protein, which resulted in higher levels of transcription from the sigma(X)-dependent promoters cinA, smf, and cglA. In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the sigma(X)-dependent cinA promoter than did the wild-type parental strain. Therefore, sigma(X) activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate sigma(X) accumulation.

Adenosine Triphosphatases↗