Mapping the σ 70 subunit contact sites on Escherichia coli RNA polymerase with a σ 70 -conjugated chemical protease

Background:No structure exists of the bacteriophage T4 activator MotA with DNA. Results: Using FeBABE, physical models, and ICM Molsoft, we determined how MotA interacts with DNA within the transcription complex. Conclusion:The unusual "double-wing" motif in MotA CTD sits within the DNA major groove. Significance: FeBABE analyses together with structures can be used to determine protein-DNA architecture within multiprotein complexes.

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confidence: 99%

Architecture of the Bacteriophage T4 Activator MotA/Promoter DNA Interaction during Sigma Appropriation

Hsieh

James

Knipling

et al. 2013

“…A cysteine substitution at position 458 had previously been constructed (22). Restriction enzymes XhoI and BamHI were used to subclone internal fragments of rpoD containing the desired mutations into pGEMD(S) (21). Construction of the other single cysteine mutants used (422C and 581C) are described elsewhere (21).…”

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confidence: 99%

“…-Mutants of rpoD encoding a single cysteine were generated by megaprimer polymerase chain reaction (19) using plasmid pGEMD(S), which contains a cysteine-free rpoD gene as a template (20,21). Mutagenic oligonucleotides complementary to the template strand of rpoD used were CYS454 (5Ј-CCATCCGTATTCCGTGTCACATGATTGAG-ACCATC-3Ј), CYS459 (5Ј-GTGCATATGATTGAGTGTATCAACAAGCTC-AAC-3Ј), and CYS461 (5Ј-TGATTGAGACCATCTGTAAGCTCAACCGTAT-T-3Ј), encoding mutations corresponding to cysteine substitutions at positions 454, 459, and 461 of 70 .…”

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confidence: 99%

Organization of Open Complexes at Escherichia coliPromoters

Bown

Owens

Meares

et al. 1999

Self Cite

A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of 70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered 70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA⅐Fe (FeBABE). RNA polymerase core reconstituted with tagged derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the ؊10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of with promoter DNA is discussed.Promoter recognition requires sequence-specific contacts by the transcriptional apparatus. At most promoters these contacts are made upstream from the transcription start point. Once the transcriptional apparatus has bound the promoter to form a closed complex, an isomerization event occurs to generate the open complex, forming the single-stranded template required for transcription. The bacterium Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription elongation with subunit composition ␣ 2 ␤␤Ј. Promoter specificity is principally afforded by a separate subunit, , which associates with the core enzyme to give holoenzyme (RNAP) 1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The 70 subunit, encoded by rpoD, is one of several subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth. RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of 70 to make stable contacts with the promoter DNA (1, 2). E. coli promoters contain two very conserved motifs, the Ϫ10 and Ϫ35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended Ϫ10 motif (5). The extended Ϫ10 motif (5Ј-TGXTATAAT-3Ј) can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within the Ϫ35 region (6, 7). Therefore this TGX motif is able to compensate for a poor Ϫ35 hexamer. The TG motif has been shown to be important for promoter activity in several other bacterial species (8 -11). Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of 70 contact the Ϫ10 and Ϫ35 hexamers, respectively (1, 2), whereas the C-terminal domain of the ␣ subunit (␣CTD) contacts the UP element (4). Recen...

“…As a control, the cysteine-less 70 holoenzyme was shown to have a specific activity similar to that of true wild type 70 enzyme under these conditions (Fig. 4) and in previously published single-and multiple-round transcription experiments (28,33). If the engineered disulfide bond restricted a critical conformational change in 70 , one would expect to see a major defect in transcriptional activity.…”

Section: Resultsmentioning

confidence: 94%

Conformational Flexibility in ς70 Region 2 during Transcription Initiation

Anthony

Burgess²

2002

Prokaryotic RNA polymerase holoenzyme is composed of core subunits (␣ 2 ␤␤) plus a factor that confers promoter specificity allowing for regulation of gene expression. Holoenzyme is known to undergo several conformational changes during the multiple steps of transcription initiation. However, the effects of these changes on the functions of specific regions have not been well characterized. In this work, we addressed the role of possible conformational change in region 2 of Escherichia coli 70 by engineering disulfide bonds that "lock" region 2.1 with region 2.2 and region 2.2 with region 2.3. When these mutant holoenzymes were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond did not result in a fundamental block, indicating that the disulfide-containing holoenzymes are active. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. Our results suggest that conformational flexibility within 70 region 2 facilitates open complex formation and transcription initiation.RNA polymerase, a multi-subunit enzyme that is made up of five polypeptide chains (␣ 2 ␤␤Ј), is solely responsible for the synthesis of messenger, transfer, and ribosomal RNA in the bacterial cell. This enzyme has two distinct functional forms: core enzyme and holoenzyme. Although core enzyme is catalytically active and capable of transcription elongation, it is unable to initiate transcription at specific promoter sequences. Promoter recognition is accomplished by binding of the specificity factor , which positions holoenzyme on the promoter sequence (1, 2). Through the use of seven factors, Escherichia coli is able to direct transcription from multiple sets of promoter sequences, which confers the ability to regulate gene expression (3, 4). Amino acid sequence alignment of factors within the 70 family identified four regions of sequence homology (regions 1-4) (5). Region 2, which contains core binding and Ϫ10 promoter recognition determinants, accounts for the most sequence similarity among 70 family members. This suggests that this region plays an important role in transcription. Because 70 region 2 was shown to be a major interaction domain with the ␤Ј coiled-coil (6 -8), this region may be the site of functionally necessary conformational changes upon interaction with core enzyme.Based upon the sum of prior mechanistic, x-ray diffraction, electron microscopy, and luminescence resonance energy transfer studies, RNA polymerase core enzyme and 70 are thought to undergo multiple conformational changes through the process of transcription initiation: from binding, to formation of the closed complex, to stable open complex formation, to transcription initiation, and finally to factor release (3, 7, 9 -13). Evidence for intermediates during this process have been identified using the well studied lacUV5 promoter (12, 14 -16). The currently accepted pathway for open complex formation based on this promoter is shown below (15).R is RNA...