Biomedical subjects

Robert L Switzer

Publications and source records attributed to Robert L Switzer.

10 recordsLinked to original sources

PubMed2006

The number of G residues in the Bacillus subtilis pyrG initially transcribed region governs reiterative transcription-mediated regulation.

Derepression of pyrG expression in Bacillus subtilis involves CTP-sensitive reiterative transcription, which introduces up to 11 extra G residues at the 5' ends of pyrG transcripts. Insertion of three or more additional Gs at the 5' end of the pyrG initially transcribed region abolished reiterative transcription and caused constitutive expression.

Bacillus subtilis↗

PubMed2005

Mutations affecting transcription pausing in the Bacillus subtilis pyr operon.

Pausing during transcription of the Bacillus subtilis pyr operon was proposed to play a role in its regulation by attenuation. Substitution mutations in the B. subtilis pyr DNA specifying the 3'-terminal nucleotides of the previously identified transcription pause sites substantially reduced pausing at these sites in vitro. This result confirms the general utility of this mutagenic strategy for studying transcriptional pausing. Pyrimidine-mediated repression in vivo of pyr-lacZ fusions containing some of these substitution mutations was substantially lower than those observed with the wild-type pyr-lacZ fusions. However, these defects in regulation were correlated with alterations in the stability of the terminator stem-loop specified by the attenuator, rather than with their effects on transcriptional pausing in vitro.

Bacillus subtilis↗

PubMed2005

Structure of the nucleotide complex of PyrR, the pyr attenuation protein from Bacillus caldolyticus, suggests dual regulation by pyrimidine and purine nucleotides.

PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60 degrees C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg2+, bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg2+ binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines.

Bacillus↗

PubMed2004

Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription.

In Bacillus subtilis and other Gram-positive bacteria, pyrimidine-mediated regulation of the pyrG gene, which encodes CTP synthetase, occurs through an attenuation mechanism involving an intrinsic transcription terminator in the pyrG leader region. Low intracellular levels of CTP prevent termination at the attenuator by a mechanism that requires the nontemplate strand sequence GGGC at the pyrG transcription initiation site (first G =+1) and the leader transcript sequence GCUCCC located at the 5' end of the terminator RNA hairpin. In this study, we demonstrate that reiterative transcription adds G residues (up to at least 10) to the 5' end of pyrG transcripts when B. subtilis cells are starved for pyrimidines but not when cells are grown with excess cytidine. Regulated repetitive addition of G residues, as well as pyrimidine-mediated pyrG regulation, requires the sequence GGGC or GGGT at the start of pyrG transcription. Mutational insertion of four extra G residues at the 5' end of the pyrG transcript (i.e., 5'-GGGGGGGC) results in constitutive pyrG expression. We propose that the incorporation of extra G residues by reiterative transcription at the wild-type promoter occurs when normal transcription elongation is stalled at position +4 by low levels of the incoming substrate, CTP, during pyrimidine limitation. The poly(G) extensions on the 5' ends of pyrG transcripts act to prevent transcription attenuation by base pairing with the sequence CUCCCUUUC located in the 5' strand of the terminator hairpin. This control mechanism is likely to operate in other Gram-positive bacteria containing similar pyrG leader sequences.

5' Untranslated Regions↗

PubMed2004

H.A. Barker.

Explore the source record for details and available documents.

Biochemistry↗

PubMed2003

Transcriptional pausing in the Bacillus subtilis pyr operon in vitro: a role in transcriptional attenuation?

The genes encoding the enzymes of pyrimidine nucleotide biosynthesis (pyr genes) are regulated in Bacillus subtilis and many other bacterial species by transcriptional attenuation. When UMP or UTP is bound to the PyrR regulatory protein, it binds to pyr mRNA at specific sequences and secondary structures in the RNA. Binding to this site prevents formation of an antiterminator stem-loop in the RNA and permits formation of a downstream terminator, leading to reduced expression of the pyr genes lying downstream from the terminator. The functioning of several other transcriptional attenuation systems has been shown to involve transcriptional pausing; this observation stimulated us to use single-round transcription of pyr genes to test for formation of paused transcripts in vitro. Using templates with each of the three known B. subtilis pyr attenuation sites, we identified one major pause site in each in which the half-life of the paused transcript was increased four- to sixfold by NusA. In each case pausing at the NusA-stimulated site prevented formation of a complete antiterminator stem-loop, while it resulted in increased time for a PyrR binding loop to form and for PyrR to bind to this loop. Thus, the pausing detected in vitro is potentially capable of playing a role in establishing the correct timing for pyr attenuation in vivo. With two of three pyr templates the combination of NusA with PyrR markedly stimulated termination of transcription at the normal termination sites. This suggests that NusA, by stabilizing pausing, plays a role in termination of pyr transcription in vivo.

Bacillus subtilis↗

PubMed2002

Kinetic studies of the uracil phosphoribosyltransferase reaction catalyzed by the Bacillus subtilis pyrimidine attenuation regulatory protein PyrR.

The PyrR protein from Bacillus subtilis and many other bacteria is a bifunctional protein. Its primary function is the regulation of expression of pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA in a uridine nucleotide-dependent manner and altering the folding of downstream RNA to promote termination of transcription. PyrR also catalyzes the uracil phosphoribosyltransferase (UPRTase) reaction even though it bears little amino acid sequence similarity to other bacterial UPRTases. The PyrR-catalyzed UPRTase reaction obeyed a Ping Pong steady state kinetic pattern under all conditions examined, but no catalysis of [(14)C]uracil-UMP and [(32)P]PP(i)-phosphoribosylpyrophosphate exchange reactions could be detected. Steady state equations for Ordered Bi Bi mechanisms for PyrR that include a kinetically irreversible conformational change after binding of PRPP but before uracil binding were shown to account for the Ping Pong pattern of the enzyme. This mechanism was supported by the following experimental observations. The reverse reaction was extremely slow with a catalytic rate constant 3300 times smaller than for the forward reaction. Patterns of product inhibition of the forward reaction were consistent with a version of the irreversible conformational change model in which PyrR returns to the unliganded conformation before dissociation of UMP and were inconsistent with several other kinetic mechanisms. UMP and phosphoribosylpyrophosphate were shown by equilibrium dialysis to bind to free PyrR (dissociation constants of 27 +/- 3 and 18 +/- 2 microm, respectively), but uracil and PP(i) did not bind at equilibrium concentrations up to 750 microm. We propose that the conformational change kinetic model developed for PyrR can also account for numerous other reports of Ping Pong kinetics for various phosphoribosyltransferases that do not form the phosphoribosyl-enzyme intermediate predicted by classic Ping Pong kinetics.

Bacillus subtilis↗

PubMed2002

cis-acting sequences of Bacillus subtilis pyrG mRNA essential for regulation by antitermination.

Expression of the Bacillus subtilis pyrG gene, which encodes CTP synthetase, is repressed by cytidine nucleotides. Regulation involves a termination-antitermination mechanism acting at a transcription terminator located within the 5' untranslated pyrG leader sequence. Deletion and substitution mutagenesis of a series of pyrG'-lacZ transcriptional fusions integrated into the B. subtilis chromosome demonstrated that only the terminator stem-loop and two specific 4- to 6-nucleotide RNA sequences were required for derepression of pyrG by starvation for cytidine nucleotides. The first sequence, GGGC/U, comprises the first four nucleotides at the 5' end of the pyrG transcript, and the second, GCUCCC, forms the first six nucleotides of the 5' strand of the terminator stem. All of the nucleotides lying between the two required RNA sequences can be deleted without loss of regulation. We propose that an as-yet-unidentified regulatory protein binds to these two RNA segments and prevents termination of transcription in the pyrG leader region when intracellular CTP levels are low.

5' Untranslated Regions↗

PubMed2002

Characterization of the interaction of Bacillus subtilis PyrR with pyr mRNA by site-directed mutagenesis of the protein.

The Bacillus subtilis PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (pyr) operon by binding in a uridine nucleotide-dependent manner to specific sites on pyr mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription. The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA. Missense mutations were constructed and evaluated for their effects on regulation of pyr genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR. A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind pyr RNA in vitro. On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding. Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly. Arg-19 was implicated in pyr regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form. The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure. The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.

Amino Acid Sequence↗

PubMed1996

The defective phosphoribosyl diphosphate synthase in a temperature-sensitive prs-2 mutant of Escherichia coli is compensated by increased enzyme synthesis.

An Escherichia coli strain which is temperature-sensitive for growth due to a mutation (prs-2) causing a defective phosphoribosyl diphosphate (PRPP) synthase has been characterized. The temperature-sensitive mutation was mapped to a 276 bp HindIII-BssHII DNA fragment located within the open reading frame specifying the PRPP synthase polypeptide. Cloning and sequencing of the mutant allele revealed two mutations. One, a G --> A transition, located in the ninth codon, was responsible for the temperature-conditional phenotype and resulted in a serine residue at this position. The wild-type codon at this position specified a glycine residue that is conserved among PRPP synthases across a broad phylogenetic range. Cells harbouring the glycine-to-serine alteration specified by a plasmid contained approximately 50% of the PRPP synthase activity of cells harbouring a plasmid-borne wild-type allele, both grown at 25 degrees C. The mutant enzyme had nearly normal heat stability, as long as it was synthesized at 25 degrees C. In contrast, there was hardly any PRPP synthase activity or anti-PRPP synthase antibody cross-reactive material present in cells harbouring the glycine to serine alteration following temperature shift to 42 degrees C. The other mutation was a C --> T transition located 39 bp upstream of the G --> A mutation, i.e. outside the coding sequence and close to the Shine-Dalgarno sequence. Cells harbouring only the C --> T mutation in a plasmid contained approximately three times as much PRPP synthase activity as a strain harbouring a plasmid-borne wild-type prs allele. In cells harbouring both mutations, the C --> T mutation appeared to compensate for the G --> A mutation by increasing the amount of a partially defective enzyme at the permissive temperature.

Alleles↗