In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing σA, the major sigma factor in growing cells

Biran, Dvora; Kroos, Lee

doi:10.1046/j.1365-2958.1997.4751843.x

Cited by 16 publications

(17 citation statements)

References 39 publications

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“…As a control, vegA expression was tested for both strains. The vegA gene is known to be expressed during vegetative growth as well as during development (26), and in vitro transcription of vegA can be initiated by RNA polymerase containing SigA, the major housekeeping sigma factor, without an additional factor (27). vegA expression was not affected by the absence of MrpC (Fig.…”

Section: Purification and Identification Of The Frua Promoter-bindingmentioning

confidence: 99%

Identification of an activator protein required for the induction of fruA , a gene essential for fruiting body development in Myxococcus xanthus

Ueki

Inouye

2003

Proc. Natl. Acad. Sci. U.S.A.

119

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Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides ؊154 and ؊107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was FruA ͉ MrpC ͉ cAMP receptor protein ͉ catabolite gene activator protein ͉ transcription activation

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Section: Purification and Identification Of The Frua Promoter-bindingmentioning

confidence: 99%

Identification of an activator protein required for the induction of fruA , a gene essential for fruiting body development in Myxococcus xanthus

Ueki

Inouye

2003

Proc. Natl. Acad. Sci. U.S.A.

119

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“…These genes or operons were originally identified by Tn5 lac insertions ⍀4403 (10), ⍀4400 (3), and ⍀4499 (9). The promoter regions do not resemble promoters that are transcribed by M. xanthus A RNA polymerase, the major vegetative RNA polymerase (2). However, both ⍀4403 and ⍀4400 have the sequence 5Ј-CATCCCT-3Ј centered at Ϫ49 bp (3).…”

mentioning

confidence: 99%

cis Elements Necessary for Developmental Expression of a Myxococcus xanthus Gene That Depends on C Signaling

Viswanathan

Kroos

2003

J Bacteriol

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Cell contact-mediated C signaling coordinates morphogenesis and gene expression during development of Myxococcus xanthus. One promoter that depends on C signaling for transcription lies upstream of ⍀4403, the site of a Tn5 lac insertion in the genome. The ⍀4403 promoter has a C-box sequence centered at ؊49 bp that matches the consensus 5-CAYYCCY-3, which is found in several C-signal-dependent promoters. Mutational analysis of the ⍀4403 promoter region was performed to test the importance of the C box and to identify other cis-acting elements. A 6-bp change in the ؊10 region eliminated promoter activity, but a 6-bp change in the ؊35 region decreased activity only about twofold. Certain single-base-pair changes in the C box centered at ؊49 bp abolished promoter activity, establishing the importance of this sequence element. Single-base-pair changes in a C-box-like sequence centered at ؊77 bp also abolished promoter activity, but the pattern of mutational effects was different from that for the C box centered at ؊49 bp. Additional single-base-pair changes indicated that all 10 bp from ؊79 to ؊70 bp are important for ⍀4403 promoter activity. Mutations at ؊59, ؊61, ؊62, and ؊63 bp also abolished promoter activity, defining a 5-bp element from ؊63 to ؊59 bp. This 5-bp element is separated from the 10-bp element (i.e., ؊79 to ؊70 bp) by 6 bp that can be changed without loss of promoter activity. Likewise, the 5 bp between the 5-bp element and the C box can be changed without loss of activity, but deletion of these 5 bp abolished activity, indicating that spacing is important. Sequences similar to the 5-and 10-bp elements, as well as the C box, are present in other C-signal-dependent promoters, suggesting some similarity in the regulatory mechanisms, but there are also indications that these cis elements do not function identically in the different promoters.

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“…Western blots employing polyclonal antibodies raised against the Bacillus subtilis RNAP holoenzyme were used to verify that the purified MxRNAP holoenzyme contained the major M. xanthus vegetative A (80 kDa; apparent size of 105 kDa), ␣ (43 kDa) and ␤/␤Ј subunits (at ϳ150 kDa). The presence of A was further confirmed using B. subtilis polyclonal anti--43 antibodies (34,35). The final MxRNAP concentration estimated using the Bio-Rad protein assay was 3.5 g/ l.…”

Section: Methodsmentioning

confidence: 99%

Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus

Lopez-Rubio

Padmanabhan

Lázaro

et al. 2004

Journal of Biological Chemistry

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The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P B ) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions ؊47 and ؊64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P B . In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P B in vitro and expression of P B in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions ؊26 to ؊40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P B -RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P B and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

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In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing σ^A, the major sigma factor in growing cells

Cited by 16 publications

References 39 publications

Identification of an activator protein required for the induction of fruA , a gene essential for fruiting body development in Myxococcus xanthus

Identification of an activator protein required for the induction of fruA , a gene essential for fruiting body development in Myxococcus xanthus

cis Elements Necessary for Developmental Expression of a Myxococcus xanthus Gene That Depends on C Signaling

Operator Design and Mechanism for CarA Repressor-mediated Down-regulation of the Photoinducible carB Operon in Myxococcus xanthus

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