scholarly journals T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 977-987 ◽  
Author(s):  
Jeanette Brill ◽  
Tamara Hoffmann ◽  
Harald Putzer ◽  
Erhard Bremer
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Northern Blot Analysis ◽  
Regulatory Mechanism ◽  
Full Length ◽  
Reporter Strain ◽  
Genetic Studies ◽  
T Box ◽  
Extension Analysis ◽  
Specific Control

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ–ProA–ProH route is responsible for the production of proline as an osmoprotectant, and the ProB–ProA–ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB–treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

scholarly journals Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity

2005 ◽  
Vol 79 (4) ◽  
pp. 2449-2460 ◽  
Author(s):  
David R. McGivern ◽  
Peter L. Collins ◽  
Rachel Fearns
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Synthesis ◽  
Northern Blot Analysis ◽  
Significant Proportion ◽  
Full Length ◽  
Mrna Synthesis ◽  
Direct Initiation ◽  
Syncytial Virus ◽  
Extension Analysis ◽  
Transcription And Replication

ABSTRACT Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3′ end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3′-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di- and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.

The ars Operon in the skinElement of Bacillus subtilis Confers Resistance to Arsenate and Arsenite

1998 ◽  
Vol 180 (7) ◽  
pp. 1655-1661 ◽  
Author(s):  
Tsutomu Sato ◽  
Yasuo Kobayashi
Keyword(s):  
Bacillus Subtilis ◽  
Constitutive Expression ◽  
Full Length ◽  
Primer Extension ◽  
Primer Extension Analysis ◽  
Negative Regulator ◽  
Arsenate Reductase ◽  
Northern Hybridization ◽  
Ars Operon ◽  
Extension Analysis

ABSTRACT The Bacillus subtilis skin element confers resistance to arsenate and arsenite. The ars operon in theskin element contains four genes in the orderarsR, ORF2, arsB, andarsC. Three of these genes are homologous to thearsR, arsB, and arsC genes from the staphylococcal plasmid pI258, while no homologs of ORF2have been found. Inactivation of arsR, arsB, orarsC results in either constitutive expression ofars, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, respectively. These results suggest that ArsR, ArsB, and ArsC function as a negative regulator, a membrane-associated protein need for extrusion of arsenite, and arsenate reductase, respectively. Expression of the arsoperon was induced by arsenate, arsenite, and antimonite. Northern hybridization and primer extension analysis showed that synthesis of a full-length ars transcript of about 2.4 kb was induced by arsenate and that the ars promoter contains sequences that resemble the −10 and −35 regions of promoters that are recognized by EςA.

scholarly journals Characterization of a New Sigma-K-Dependent Peptidoglycan Hydrolase Gene That Plays a Role in Bacillus subtilis Mother Cell Lysis

1999 ◽  
Vol 181 (20) ◽  
pp. 6230-6237 ◽  
Author(s):  
Fajar Aji Nugroho ◽  
Hiroki Yamamoto ◽  
Yasuo Kobayashi ◽  
Junichi Sekiguchi
Keyword(s):  
Bacillus Subtilis ◽  
Mother Cell ◽  
Northern Blot Analysis ◽  
Cell Lysis ◽  
Peptidoglycan Hydrolase ◽  
Double Mutation ◽  
Optimum Ph ◽  
High Level ◽  
Extension Analysis

ABSTRACT Bacillus subtilis produces a 30-kDa peptidoglycan hydrolase, CwlH, during the late sporulation phase. Disruption ofyqeE led to a complete loss of CwlH formation, indicating the identity of yqeE with cwlH. Northern blot analysis of cwlH revealed a 0.8-kb transcript after 6 to 7.5 h for the wild-type strain but not for the ςF, ςE, ςG, and ςK mutants. Expression of the ςK-dependent cwlH gene depended on gerE. Primer extension analysis also suggested that cwlH is transcribed by EςK RNA polymerase. CwlH produced in Escherichia coli harboring acwlH plasmid is anN-acetylmuramoyl-l-alanine amidase (EC3.5.1.28 ) and exhibited an optimum pH of 7.0 and high-level binding to the B. subtilis cell wall. A cwlC cwlH double mutation led to a lack of mother cell lysis even after 7 days of incubation in DSM medium, but the single mutations led to mother cell lysis after 24 h.

scholarly journals Destabilization of Bacteriophage T4 mRNAs by a Mutation of Gene 61.5

Genetics ◽  
1996 ◽  
Vol 144 (1) ◽  
pp. 7-14 ◽  
Author(s):  
Toshie Kai ◽  
Harold E Selick ◽  
Tetsuro Yonesaki
Keyword(s):  
Northern Blot Analysis ◽  
Bacteriophage T4 ◽  
Decay Rates ◽  
Full Length ◽  
Late Gene ◽  
Transcription Start ◽  
Endonucleolytic Cleavage ◽  
Late Genes ◽  
Infected Cells ◽  
Extension Analysis

Abstract We identified a novel gene of bacteriophage T4, gene 61.5, which appears to be involved in protein synthesis late in infection. Northern blot analysis revealed that a mutant of 61.5 accumulated truncated transcripts of representative late genes. Using a double mutant of genes 61.5 and 55, which prevents transcription of late genes, we demonstrate that even transcripts of middle genes, while full-length when initially expressed, are similarly truncated at later stages of infection. These results indicate that the abnormality in transcript length occurs late in infection, regardless of whether the transcript derives from a middle or a late gene. Primer-extension analysis revealed that the 5′ ends of the late gene 23 transcripts that accumulated in gene 61.5 mutant-infected cells were located at internal discrete sites as well as at the expected transcription start site. Moreover, the decay rates of full-length transcripts from genes uvsY or 45 were more than twofold faster in the absence of a functional gene 61.5. These results suggest that mutation of gene 61.5 activates endonucleolytic cleavage of middle and late transcripts, probably by RNase M.

scholarly journals Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate

1972 ◽  
Vol 129 (2) ◽  
pp. 367-372 ◽  
Author(s):  
E. Komárková ◽  
J. Hradec
Keyword(s):  
Protein Synthesis ◽  
Liver Tissue ◽  
Regulatory Mechanism ◽  
Relative Content ◽  
Time Intervals ◽  
Peptidyl Transfer ◽  
Significant Stimulation ◽  
Peptide Elongation

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.

scholarly journals Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

2004 ◽  
Vol 186 (16) ◽  
pp. 5392-5399 ◽  
Author(s):  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Assay System ◽  
Nascent Transcript ◽  
Vitro Assay ◽  
T Box ◽  
Transcription Antitermination ◽  
Transcriptional Pausing ◽  
Transcription Complexes ◽  
Kinetics Of

ABSTRACT Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure

1993 ◽  
Vol 13 (8) ◽  
pp. 4826-4835
Author(s):  
C L Hsu ◽  
A Stevens
Keyword(s):  
Full Length ◽  
Yeast Cells ◽  
Mrna Turnover ◽  
Turnover Rates ◽  
Wild Type ◽  
Mrna Species ◽  
Turnover Process ◽  
Hydrolysis Of ◽  
Extension Analysis ◽  
Wild Type Yeast

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

The program of protein synthesis during sporulation in Bacillus subtilis

Cell ◽  
1976 ◽  
Vol 8 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Thomas Linn ◽  
Richard Losick
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis

scholarly journals Genetic Studies of Leucine Biosynthesis in Bacillus subtilis

1973 ◽  
Vol 116 (2) ◽  
pp. 719-726 ◽  
Author(s):  
Jonathan B. Ward ◽  
Stanley A. Zahler
Keyword(s):  
Bacillus Subtilis ◽  
Leucine Biosynthesis ◽  
Genetic Studies
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