HO.bul. and DNase I Probing of E.sigma.70 RNA Polymerase-.lambda.PR Promoter Open Complexes: Mg2+ Binding and Its Structural Consequences at the Transcription Start Site

Craig, Maria; Suh, Won-Chul; Record, M. Thomas

doi:10.1021/bi00048a004

Cited by 100 publications

(141 citation statements)

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“…We suggest this is in part due to the location of Ler binding, between positions 2221 and 260, and thus it is probable that this regulatory protein interacts with RNA polymerase, which binds to s 70 -dependent promoter regions extending from positions~255 to +20 in relation to the start of transcription (Craig et al, 1995). Perhaps consistent with this prediction, we have been unable to identify an additional negative-acting factor for LEE5 by using genetic approaches (data not shown).…”

Section: Discussionmentioning

confidence: 67%

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

et al. 2011

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Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoidassociated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler-DNA structures that are predicted to be involved in stimulating gene expression.

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Section: Discussionmentioning

confidence: 67%

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

et al. 2011

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“…Surprisingly, protection of DNA in I 1 from DNase I cleavage extends upstream only to Ϫ52 (template) on ''full-length'' P R , similar to what is observed in RP o (6). Because DNase I might displace weak upstream interactions in I 1 (8,9), other techniques are required to probe RNAP-DNA contacts in this transient intermediate. Sclavi et al (10) recently reported an elegant ''real-time'' footprinting study of RP o formation at the T7A1 promoter using x-ray-generated ⅐OH and rapid quench mixing.…”

mentioning

confidence: 70%

“…Additionally, the rapid rate of ⅐OH reaction with the DNA backbone means that fractional protection is approximately proportional to fractional occupancy for a short-lived, rapidly equilibrating intermediate like I 1 (10). By following the appearance of protection of the DNA backbone as a function of time (milliseconds to seconds) after mixing at 37°C, this study (10) circumvented potential issues raised by trapping promoter complexes at low temperature (11) and possible displacement of weak upstream interactions by DNase I (8,9).…”

mentioning

confidence: 99%

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Davis¹,

Bingman

Landick³

et al. 2007

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

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197

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initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

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“…Studies in the E. coli enzyme suggest that this simple question can be complicated by the existence of multiple forms of open and closed complexes (31,41). It is tempting to use the fluorescence changes associated with DNA binding in a quantitative sense to determine the thermodynamic distribution between open and closed states.…”

Section: Estimation Of Fractional Meltingmentioning

confidence: 99%

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

et al. 2007

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T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein.Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the Cterminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

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HO.bul. and DNase I Probing of E.sigma.70 RNA Polymerase-.lambda.PR Promoter Open Complexes: Mg2+ Binding and Its Structural Consequences at the Transcription Start Site

Cited by 100 publications

References 51 publications

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

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