Promoter opening via a DNA fork junction binding activity

Guo, Yanjie; Gralla, Jay D.

doi:10.1073/pnas.95.20.11655

Cited by 95 publications

(167 citation statements)

References 39 publications

Supporting

Mentioning

153

Contrasting

Order By: Relevance

“…To form stable RNAP-DNA complexes, we used a fork-junction template (6,25), comprising the vapB10 promoter (VFJ-28) (Figs. 4A and S3A) along with an anticonsensus control (VFJ28anti) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Hubin

Tabib-Salazar

Humphrey

et al. 2015

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

View full text Add to dashboard Cite

Gene expression is highly regulated at the step of transcription initiation, and transcription activators play a critical role in this process. RbpA, an actinobacterial transcription activator that is essential in Mycobacterium tuberculosis (Mtb), binds selectively to group 1 and certain group 2 σ-factors. To delineate the molecular mechanism of RbpA, we show that the Mtb RbpA σ-interacting domain (SID) and basic linker are sufficient for transcription activation. We also present the crystal structure of the Mtb RbpA-SID in complex with domain 2 of the housekeeping σ-factor, σ A . The structure explains the basis of σ-selectivity by RbpA, showing that RbpA interacts with conserved regions of σ A as well as the nonconserved region (NCR), which is present only in housekeeping σ-factors. Thus, the structure is the first, to our knowledge, to show a protein interacting with the NCR of a σ-factor. We confirm the basis of selectivity and the observed interactions using mutagenesis and functional studies. In addition, the structure allows for a model of the RbpA-SID in the context of a transcription initiation complex. Unexpectedly, the structural modeling suggests that RbpA contacts the promoter DNA, and we present in vivo and in vitro studies supporting this finding. Our combined data lead to a better understanding of the mechanism of RbpA function as a transcription activator.RbpA | X-ray crystallography | transcription | actinobacteria | mycobacteria

show abstract

Section: Resultsmentioning

confidence: 99%

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Hubin

Tabib-Salazar

Humphrey

et al. 2015

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

View full text Add to dashboard Cite

show abstract

“…The closed complex has base pair Ϫ11 transiently melted (12). The enzyme has a high affinity for DNA containing such double-strand/ single-strand fork junction structures (8,9). When artificial fork probes are used that mimic parts of this structure, the affinity is highest when the bottom strand unpaired nucleotide is present (Fig.…”

Section: Resultsmentioning

confidence: 99%

Roles for the C-terminal Region of Sigma 54 in Transcriptional Silencing and DNA Binding

Wang

Gralla

2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Twenty-one conserved positively charged and aromatic amino acids between residues 331 and 462 of sigma 54 were changed to alanine, and the mutant proteins were studied by transcription, band shift analysis, and footprinting in vitro. A small segment corresponding to the rpoN box was found to be most important for binding duplex DNA. Two amino acids, 52 residues apart, were found to be critical for maintaining transcriptional silencing in the absence of activator. These two activator bypass mutants and several other mutants failed to bind the type of fork junction DNA thought to be required to maintain silencing. The two bypass mutants showed a binding pattern to DNA probes that was unique, both in comparison to other C-terminal mutants and to previously known N-terminal bypass mutants. On this basis, a model is proposed for the role of the C terminus and the N terminus of sigma 54 in enhancerdependent transcription.

show abstract

“…1 was investigated. These are partial promoters, designed to mimic the conformation of DNA for intermediates in the process of open complex formation (20,26). Nucleation of promoter DNA melting (the I 1 to I 2 step) likely involves forcing the Ϫ11A out of the DNA helix and into a pocket on the RNAP ("base flipping") with concomitant acquisition of stability to heparin challenge (10,27).…”

Section: Determination Of Strand Opening By Kmno 4 Probing: Correctiomentioning

confidence: 99%

Mechanistic Differences in Promoter DNA Melting by Thermus aquaticus and Escherichia coli RNA Polymerases

Schroeder

deHaseth

2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the A subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75°C. However, homologous substitutions in 70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37°C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations. Transcription in bacteria is catalyzed by DNA-dependent RNA polymerase (RNAP), 1 a key enzyme for gene expression and its regulation (1, 2). Specific recognition of promoter DNA is mediated by an initiation subunit (sigma factor). The functional RNAP "holoenzyme" results when the sigma factor binds to the catalytic component, the "core" enzyme. A highly conserved region of 18 amino acids (designated region 2.3) of the main bacterial sigma factors has been shown to play a role in the melting process (3-6). Aromatic amino acid side chains in this region are positioned on the same side of an ␣ helix, poised to interact with the exposed bases of the promoter DNA (7,8).Studies with Escherichia coli (Eco) RNAP have indicated that upon RNAP binding to promoter DNA, an unstable closed complex (RP c ) is formed, followed by at least two additional intermediates (I 1 and I 2 ) before the initiation-competent, stable open complex (RP o ) is formed (9, 10), in which a 14-bp region of the promoter DNA, including the start site of transcription (2), has been melted. R ϩ P 7 RP c 7 I 1 7 I 2 7 RP o SCHEME 1In addition to the promoter DNA, the RNAP is thought to undergo conformational changes as well (9, 10).The past 6 years have seen a dramatic increase in structural information for bacterial RNAP, primarily because of the successful crystallization of the RNAP from two thermophilic bacteria, Thermus aquaticus (Taq) and Thermus thermophilus (8,(11)(12)(13)(14). The RNAP from these two organisms share extensive homology with all subunits of Eco RNAP, including the primary sigma factors. Four amino acids are different between the DNA melting regions (2.3) of Taq A and Eco 70 , but in three of the four cases the differences involve amino acids with chemically similar side chains. It is not well understood to what extent the mechanism of...

show abstract

Promoter opening via a DNA fork junction binding activity

Cited by 95 publications

References 39 publications

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Structural, functional, and genetic analyses of the actinobacterial transcription factor RbpA

Roles for the C-terminal Region of Sigma 54 in Transcriptional Silencing and DNA Binding

Mechanistic Differences in Promoter DNA Melting by Thermus aquaticus and Escherichia coli RNA Polymerases

Contact Info

Product

Resources

About