scholarly journals The −10 Region Is a Key Promoter Specificity Determinant for the Bacillus subtilis Extracytoplasmic-Function ς Factors ςX and ςW

2001 ◽  
Vol 183 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Jian Qiu ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Context Effects ◽  
Promoter Strength ◽  
Binding Properties ◽  
Promoter Specificity ◽  
Dna Binding Properties

ABSTRACT Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in Bacillus subtilis which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ςX and ςW factors can be explained by overlapping specificity for the −10 region: ςX recognizes −10 elements with the sequence CGAC and ςW recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ςX-specific autoregulatory site (PX), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ςW autoregulatory site (PW) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ςX to the ςW regulon or, conversely, from the ςW to the ςX regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ςW.

scholarly journals Properties of Bacillus subtilis σA Factors with Region 1.1 and the Conserved Arg-103 at the N Terminus of Region 1.2 Deleted

2004 ◽  
Vol 186 (8) ◽  
pp. 2366-2375 ◽  
Author(s):  
Hsin-Hsien Hsu ◽  
Wei-Cheng Huang ◽  
Jia-Perng Chen ◽  
Liang-Yin Huang ◽  
Chai-Fong Wu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Binding Activity ◽  
Transcription Activity ◽  
N Terminus ◽  
Σ Factor

ABSTRACT σ factors in the σ70 family can be classified into the primary and alternative σ factors according to their physiological functions and amino acid sequence similarities. The primary σ factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative σ factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis σA, which belongs to a subgroup of the primary σ factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of σA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated σA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of σA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the σA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated σA and greatly reduced the transcription activity of the truncated σA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of σA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length σA in RNA polymerase.

scholarly journals Termination-altering mutations in the second-largest subunit of yeast RNA polymerase III.

1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
The Other ◽  
Polymerase Iii

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.

scholarly journals Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU

2020 ◽  
Vol 21 (24) ◽  
pp. 9553
Author(s):  
Szu-Ning Lin ◽  
Gijs J.L. Wuite ◽  
Remus T. Dame
Keyword(s):  
Local Concentration ◽  
Complex Interplay ◽  
Binding Properties ◽  
In Vitro System ◽  
Hu Protein ◽  
Tethered Particle Motion ◽  
Bacterial Nucleoid ◽  
Dna Binding Properties

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

scholarly journals Characterization of LrpC DNA-Binding Properties and Regulation of Bacillus subtilis lrpC Gene Expression

2000 ◽  
Vol 182 (16) ◽  
pp. 4414-4424 ◽  
Author(s):  
Christophe Beloin ◽  
Rachel Exley ◽  
Anne-Laure Mahé ◽  
Mohamed Zouine ◽  
Stephanie Cubasch ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Biological Significance ◽  
Stationary Growth Phase ◽  
Upstream Region ◽  
Binding Properties ◽  
Sequencing Project ◽  
Dna Binding Properties ◽  
Transcriptional Fusions

ABSTRACT The lrpC gene was identified during the Bacillus subtilis genome sequencing project. Previous experiments suggested that LrpC has a role in sporulation and in the regulation of amino acid metabolism and that it shares features withEscherichia coli Lrp, a transcription regulator (C. Beloin, S. Ayora, R. Exley, L. Hirschbein, N. Ogasawara, Y. Kasahara, J. C. Alonso, and F. Le Hégarat, Mol. Gen. Genet. 256:63–71, 1997). To characterize the interactions of LrpC with DNA, the protein was overproduced and purified. We show that LrpC binds to multiple sites in the upstream region of its own gene with a stronger affinity for a region encompassing P1, one of the putative promoters identified (P1 and P2). By analyzing lrpC-lacZ transcriptional fusions, we demonstrated that P1 is the major in vivo promoter and that, unlike many members of the lrp/asnC family,lrpC is not negatively autoregulated but rather slightly positively autoregulated. Production of LrpC in vivo is low in both rich and minimal media (50 to 300 LrpC molecules per cell). In rich medium, the cellular LrpC content is six- to sevenfold lower during the exponentional phase than during the stationary growth phase. Possible determinants and the biological significance of the regulation oflrpC expression are discussed.

scholarly journals Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

2002 ◽  
Vol 184 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
In Vitro Transcription ◽  
Dna Binding Domain ◽  
Promoter Activation ◽  
Transcription Activators

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

scholarly journals Mechanism of Transcription Activation at the comG Promoter by the Competence Transcription Factor ComK of Bacillus subtilis

2004 ◽  
Vol 186 (4) ◽  
pp. 1120-1128 ◽  
Author(s):  
K. A. Susanna ◽  
A. F. van der Werff ◽  
C. D. den Hengst ◽  
B. Calles ◽  
M. Salas ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Complex Signal ◽  
Type I ◽  
Binding Characteristics ◽  
Α Subunits

ABSTRACT The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding and uptake machinery and of genes required for homologous recombination. In vivo and in vitro experiments have shown that ComK is responsible for transcription activation at the comG promoter. In this study, we investigated the mechanism of this transcription activation. The intrinsic binding characteristics of RNA polymerase with and without ComK at the comG promoter were determined, demonstrating that ComK stabilizes the binding of RNA polymerase to the comG promoter. This stabilization probably occurs through interactions with the upstream DNA, since a deletion of the upstream DNA resulted in an almost complete abolishment of stabilization of RNA polymerase binding. Furthermore, a strong requirement for the presence of an extra AT box in addition to the common ComK-binding site was shown. In vitro transcription with B. subtilis RNA polymerase reconstituted with wild-type α-subunits and with C-terminal deletion mutants of the α-subunits was performed, demonstrating that these deletions do not abolish transcription activation by ComK. This indicates that ComK is not a type I activator. We also show that ComK is not required for open complex formation. A possible mechanism for transcription activation is proposed, implying that the major stimulatory effect of ComK is on binding of RNA polymerase.

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