Tunable transcription factor library for robust quantification of regulatory properties in Escherichia coli.

Predicting the quantitative regulatory function of transcription factors (TFs) based on factors such as binding sequence, binding location, and promoter type is not possible. The interconnected nature of gene networks and the difficulty in tuning individual TF concentrations make the isolated study of TF function challenging. Here, we present a library of Escherichia coli strains designed to allow for precise control of the concentration of individual TFs enabling the study of the role of TF concentration on physiology and regulation. We demonstrate the usefulness of this resource by measuring the regulatory function of the zinc-responsive TF, ZntR, and the paralogous TF pair, GalR/GalS. For ZntR, we find that zinc alters ZntR regulatory function in a way that enables activation of the regulated gene to be robust with respect to ZntR concentration. For GalR and GalS, we are able to demonstrate that these paralogous TFs have fundamentally distinct regulatory roles beyond differences in binding affinity.

Analysis of mRNA Decay Intermediates in Bacillus subtilis 3' Exoribonuclease and RNA Helicase Mutant Strains.

The Bacillus subtilis genome encodes four 3' exoribonucleases: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Previous work showed that PNPase, encoded by the pnpA gene, is the major 3' exonuclease involved in mRNA turnover; in a pnpA deletion strain, numerous mRNA decay intermediates accumulate. Whether B. subtilis mRNA decay occurs in the context of a degradosome complex is controversial. In this study, global mapping of mRNA decay intermediate 3' ends within coding sequences was performed in strains that were either deleted for or had an inactivating point mutation in the pnpA gene. The patterns of 3'-end accumulation in these strains were highly similar, which may have implications for the role of a degradosome in mRNA decay. A comparison with mapped 3' ends in a strain lacking CshA, the major RNA helicase, indicated that many mRNAs require both PNPase and CshA for efficient decay. Transcriptome sequencing (RNA-seq) analysis of strains lacking RNase R suggested that this enzyme did not play a major role in mRNA turnover in the wild-type strain. Strains were constructed that contained only one of the four known 3' exoribonucleases. When RNase R was the only 3' exonuclease present, it was able to degrade a model mRNA efficiently, showing processive decay even through a strong stem-loop structure that inhibits PNPase processivity. Strains containing only RNase PH or only YhaM were also insensitive to this RNA secondary structure, suggesting the existence of another, as-yet-unidentified, 3' exoribonuclease. IMPORTANCE The ability to rapidly change bacterial gene expression programs in response to environmental conditions is highly dependent on the efficient turnover of mRNA. While much is known about the regulation of gene expression at the transcriptional and translational levels, much less is known about the intermediate step of mRNA decay. Here, we mapped the 3' ends of mRNA decay intermediates in strains that were missing the major 3' exoribonuclease PNPase or the RNA helicase CshA. We also assessed the roles of three other B. subtilis 3' exonucleases in the mRNA decay process. The data confirm the primary role of PNPase in mRNA turnover and suggest the involvement of one or more unknown RNases.

Discovery and initial characterization of YloC, a novel endoribonuclease in Bacillus subtilis.

The Bacillus subtilis genome is predicted to encode numerous ribonucleases, including four 3' exoribonucleases that have been characterized to some extent. A strain containing gene knockouts of all four known 3' exoribonucleases is viable, suggesting that one or more additional RNases remain to be discovered. A protein extract from the quadruple RNase mutant strain was fractionated and RNase activity was followed, resulting in the identification of an enzyme activity catalyzed by the YloC protein. YloC is an endoribonuclease and is a member of the highly conserved "YicC family" of proteins that is widespread in bacteria. YloC is a metal-dependent enzyme that catalyzes the cleavage of single-stranded RNA, preferentially at U residues, and exists in an oligomeric form, most likely a hexamer. As such, YloC shares some characteristics with the SARS-CoV Nsp15 endoribonuclease. While the in vivo function of YloC in B. subtilis is yet to be determined, YloC was found to act similarly to YicC in an Escherichia coli in vivo assay that assesses decay of the small RNA, RyhB. Thus, YloC may play a role in small RNA regulation.

Quantifying the regulatory role of individual transcription factors in Escherichia coli.

Gene regulation often results from the action of multiple transcription factors (TFs) acting at a promoter, obscuring the individual regulatory effect of each TF on RNA polymerase (RNAP). Here we measure the fundamental regulatory interactions of TFs in E. coli by designing synthetic target genes that isolate individual TFs' regulatory effects. Using a thermodynamic model, each TF's regulatory interactions are decoupled from TF occupancy and interpreted as acting through (de)stabilization of RNAP and (de)acceleration of transcription initiation. We find that the contribution of each mechanism depends on TF identity and binding location; regulation immediately downstream of the promoter is insensitive to TF identity, but the same TFs regulate by distinct mechanisms upstream of the promoter. These two mechanisms are uncoupled and can act coherently, to reinforce the observed regulatory role (activation/repression), or incoherently, wherein the TF regulates two distinct steps with opposing effects.

Polynucleotide phosphorylase and RNA helicase CshA cooperate in Bacillus subtilis mRNA decay.

Polynucleotide phosphorylase (PNPase), a 3' exoribonuclease that degrades RNA in the 3'-to-5' direction, is the major mRNA decay activity in Bacillus subtilis. PNPase is known to be inhibited in vitro by strong RNA secondary structure, and rapid mRNA turnover in vivo is thought to require an RNA helicase activity working in conjunction with PNPase. The most abundant RNA helicase in B. subtilis is CshA. We found for three small, monocistronic mRNAs that, for some RNA sequences, PNPase processivity was unimpeded even without CshA, whereas others required CshA for efficient degradation. A novel colour screen for decay of mRNA in B. subtilis was created, using mRNA encoded by the slrA gene, which is degraded from its 3' end by PNPase. A significant correlation between the predicted strength of a stem-loop structure, located in the body of the message, and PNPase processivity was observed. Northern blot analysis confirmed that PNPase processivity was greatly hindered by the internal RNA structure, and even more so in the absence of CshA. Three other B. subtilis RNA helicases did not appear to be involved in mRNA decay during vegetative growth. The results confirm the hypothesis that efficient 3' exonucleolytic decay of B. subtilis RNA depends on the combined activity of PNPase and CshA.

Inefficient translation of nsrR constrains behaviour of the NsrR regulon in Escherichia coli.

The NsrR protein of Escherichia coli is a transcriptional repressor that contains an [Fe-S] cluster that is the binding site for nitric oxide (NO). Reaction of NsrR with NO leads to de-repression of its target genes, which include those encoding an NO scavenging flavohaemoglobin and the RIC (repair of iron centres) protein involved in the repair of NO-damaged [Fe-S] clusters. The nsrR gene is promoter proximal in a transcription unit with rnr, encoding the cold shock-inducible RNase R. Here, we show that nsrR is expressed from a strong promoter, but that its translation is extremely inefficient, leading to a low cellular NsrR concentration. Conversion of the nsrR start codon from the wild-type GUG to AUG increased the efficiency of translation (which, nevertheless, remained extremely low) and had measurable effects on the expression of some NsrR-regulated genes. We conclude that NsrR abundance in the cell is such that promoters with low-affinity NsrR binding sites may partially escape NsrR-mediated repression. Expression profiling confirmed that genes regulated by NsrR (whether directly or indirectly) tend to express lower mRNA levels when the nsrR start codon is AUG than when it is GUG. Transcriptomics data implicated the pyruvate oxidase gene poxB as a novel NsrR target, which we confirmed and showed to be due to read-through transcription from the upstream hcp-hcr genes. We also present evidence to suggest that NsrR is a regulator of the sufABCDSE genes, which encode the components of an [Fe-S] cluster biogenesis and repair system.

Genotype of the LMNA 1908C>T variant is associated with generalized obesity in Asian Indians in North India.

OBJECTIVE: To determine the frequency distribution of LMNA 1908C>T SNP and its association with generalized obesity, abdominal obesity and coexistent metabolic disorders in nondiabetic Asian Indians living in a metropolitan city of north India. DESIGN: A cross-sectional population-based study of LMNA 1908C>T polymorphism with obesity and insulin resistance as outcome. PATIENTS: Five hundred and fifty-one Asian Indians, with 240 obese and 289 nonobese subjects. MEASUREMENTS: Allelic and genotypic frequency of LMNA 1908C>T were determined by PCR-RFLP. Association of LMNA alleles and genotypes was analysed with various measures of obesity [BMI ≥ 25 kg/m(2) , percentage body fat (by DEXA); subcutaneous and intra-abdominal fat at L2-3 level by single slice MRI in a subsample and surrogate marker of insulin resistance (fasting serum insulin levels >10 µU/ml in men and >11 µU/ml in women). RESULTS: Forty-six per cent of the subjects had generalized obesity while 54% had abdominal obesity. Frequency of C and T alleles was 0·71 and 0·29, respectively. Higher frequency of variant allele (T) was observed in obese than nonobese individuals (P = 0·001). On multivariate analysis adjusting for age, gender and serum insulin levels, subjects with LMNA1908T/T genotype were at 5·6 times higher risk [OR (95% CI): 5·6 (2·5-12·2), P = 0·001], while individuals with genotypes with at least one T allele, i.e. 1908C/T and T/T genotypes, were at 2·7 times higher odds to develop generalized obesity [OR (95% CI): 2·7 (1·8-4·1), P = 0·001]. CONCLUSION: LMNA 1908T/T and C/T genotypes emerged as independent genetic risk factors for generalized obesity in Asian north Indians.