The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript or template DNA onto x-ray crystal structure. In resulting model, downstream duplex is nestled in trough formed β′ subunit enclosed on top β subunit. RNAP channel, RNA/DNA hybrid extends from enzyme active site, along region harboring rifampicin resistance mutations, to “rudder.” single-stranded then extruded another channel...

Coliphage HK022 Nun blocks superinfection by coliphage λ stalling RNA polymerase (RNAP) translocation specifically on DNA. To provide a structural framework to understand how RNAP translocation, we determined structures of Escherichia coli ternary elongation complexes (TECs) with and without single-particle cryo-electron microscopy. fits tightly into the TEC taking advantage gaps between nucleic acids. The C-terminal segment interacts β β’ subunits inside active site cleft as well nearly...

When the Mg 2+ ion in catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe , hydroxyl radicals are generated. In promoter complex, such cleave template DNA near transcription start site, whereas β′ subunit cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution three aspartate residues alanine creates dominant lethal mutation. The mutant RNAP catalytically inactive but can bind promoters and form an open complex. fails to support -induced...

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing end and backtracks DNA template. Prokaryotic Gre factors rescue backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes disengaged 3' segment. By using RNA-protein crosslinking in defined complexes, site-directed mutagenesis, discriminative biochemical assays, docking two protein structures, we show that acts by providing carboxylate...

RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs reactive substituents at terminal phosphate. This treatment followed by addition a pyrimidine [alpha-32P]NTP. Due to 'catalytic competence' some residues reagents bound covalently near active center first stage, active-center-catalyzed synthesis phosphodiester bond occurred, radioactive general formula -pNpN (where p = phosphate) appeared attached enzyme....

A method is proposed for localization of the sites affinity labelling β subunit Escherichia coli RNA polymerase. The principle this similar to that methods rapid sequencing nucleic acids. polypeptide bearing a radioactive label at one amino acid residues subjected short‐term treatment with cyanogen bromide. conditions reaction are selected in such way less than cleavage occurs on average per chain. Two series peptides formed, involving all possible N‐terminal and other C‐terminal peptides....

Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent the RNAP active center and preventing synthesis of products >2-3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351-363] proposed that rifamycins function allosteric modulation Mg(2+) presented three lines biochemical evidence consistent with this proposal. Here, we show do not affect affinity center, reassess evidence, obtaining results supportive We conclude center.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by productive elongation complex. Two these beta subunit and one is beta' subunit. During elongation, transcription complex occasionally undergoes an arrest whereby it can neither extend nor release transcript. It demonstrated arrested complex, contacts lost, while new contact becomes prominent. Thus, appears to involve...

The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in beta subunit Escherichia coli enzyme (Lys1065 His1237) were mapped close to binding site priming reaction. Surprisingly, mutational substitution these residues (Lys1065----Arg His1237----Ala) did not inactivate catalytic function, but inhibited transition from initiation...

Novel amino-reactive derivatives of lanthanide-based luminescent labels enhanced brightness and metal retention were synthesized used for the detection cDNA oligonucleotides by molecular beacons. Time-resolved acquisition signal that occurs upon hybridization probe to target enabled avoidance short-lived background fluorescence, markedly enhancing sensitivity detection, which was less than 1 pM. This value is about 50 100 times more sensitive level achieved with conventional...

Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the wild-type cells, lower. Addition a 3-amino group 2,4-dione core increased relative while...

Widespread fluoroquinolone resistance has drawn attention to quinazolinediones (diones), fluoroquinolone-like topoisomerase poisons that are unaffected by common quinolone-resistance mutations. To better understand differences between quinolones and diones, we examined their impact on the formation of cleaved complexes (drug-topoisomerase-DNA in which DNA moiety is broken) with gyrase, one two bacterial targets drugs. Formation complexes, measured linearization a circular substrate, required...

During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2–8 nt in length before disengaging from promoter isomerizing to a stable elongation complex. In this study, we used RNA⋅protein RNA⋅DNA crosslinking methods probe location newly synthesized halted complexes. The results indicate remains hybrid for about 8 site nucleotide addition emerges surface enzyme 12 site. Strikingly, as transcript leaves its with...

The Fe 2+ ion that specifically replaces Mg in the active center of RNA polymerase generates reactive hydroxyl radicals cause highly localized cleavage polypeptide chains. Mapping sites revealed overall architecture center. Nine distinct sites, five β subunit and four β′ Escherichia coli polymerase, all at or near conserved sequence motifs, are brought together enzyme’s ternary structure within distance ≈1 nm from Me . These located least six different domains subunits, reflecting modular...