Alex Goldfarb

Structural Mechanism for Rifampicin Inhibition of Bacterial RNA Polymerase

Rifampicin (Rif) is one of the most potent and broad spectrum antibiotics against bacterial pathogens and is a key component of anti-tuberculosis therapy, stemming from its inhibition of the bacterial RNA polymerase (RNAP). We determined the crystal structure of Thermus aquaticus core RNAP complexed with Rif. The inhibitor binds in a pocket of the RNAP beta subunit deep within the DNA/RNA channel, but more than 12 A away from the active site. The structure, combined with biochemical results, explains the effects of Rif on RNAP function and indicates that the inhibitor acts by directly blocking the path of the elongating RNA when the transcript becomes 2 to 3 nt in length.

Transcription processivity: protein-DNA interactions holding together the elongation complex.

The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the β′ subunit and the R interaction, to the COOH-terminal catalytic domain of the β subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity.

Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase.

In DNA‐dependent RNA polymerases, reactions of RNA synthesis and degradation are performed by the same active center (in contrast to DNA polymerases in which they are separate). We propose a unified catalytic mechanism for multisubunit RNA polymerases based on the analysis of its 3′–5′ exonuclease reaction in the context of crystal structure. The active center involves a symmetrical pair of Mg2+ ions that switch roles in synthesis and degradation. One ion is retained permanently and the other is recruited ad hoc for each act of catalysis. The weakly bound Mg2+ is stabilized in the active center in different modes depending on the type of reaction: during synthesis by the β,γ‐phosphates of the incoming substrate; and during hydrolysis by the phosphates of a non‐base‐paired nucleoside triphosphate. The latter mode defines a transient, non‐specific nucleoside triphosphate‐binding site adjacent to the active center, which may serve as a gateway for polymerization of substrates.

Mapping of Catalytic Residues in the RNA Polymerase Active Center

When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the β′ subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutation. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex. The mutant fails to support Fe2+-induced cleavage of DNA or protein. Thus, the NADFDGD motif is involved in chelation of the active center Mg2+.

Coupling between transcription termination and RNA polymerase inchworming

Advancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism.

Donation of catalytic residues to RNA polymerase active center by transcription factor Gre

During transcription elongation, RNA polymerase (RNAP) occasionally loses its grip on the growing RNA end and backtracks on the DNA template. Prokaryotic Gre factors rescue the backtracked ternary elongating complex through stimulation of an intrinsic endonuclease activity, which removes the disengaged 3′ RNA segment. By using RNA-protein crosslinking in defined ternary elongating complexes, site-directed mutagenesis, discriminative biochemical assays, and docking of the two protein structures, we show that Gre acts by providing two carboxylate residues for coordination of catalytic Mg2+ ion in the RNAP active center. A similar mechanism is suggested for the functionally analogous eukaryotic SII factor. The results expand the general two-metal model of RNAP catalytic mechanism whereby one of the Mg2+ ions is permanently retained, whereas the other is recruited ad hoc by an auxiliary factor.

Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase.

Publisher Summary This chapter discusses the use of histidine (His) tags for obtaining transcription intermediates. The mechanism and regulation of transcription depends largely on the development of experimental techniques permitting dissection of the multistep transcriptional cycle. The His tag technology has been applied for the study of mechanisms of elongation, pausing, factor-independent termination, and interaction of RNA polymerase (RNAP) with transcriptional factors. In addition, histidine tags have been used for the rapid purification of RNAP from cells, in vitro reconstitution of RNAP from individually expressed subunits, screening of genetically engineered RNAP mutations, identification of specific fragments among the products of partial proteolysis, and probing of the surface of a RNAP molecule. The chapter discusses the preparation of His-tagged RNAP from cells. Solid-phase transcription with His-tagged RNAP is also given.

Structural Modules of the Large Subunits of RNA Polymerase INTRODUCING ARCHAEBACTERIAL AND CHLOROPLAST SPLIT SITES IN THE β AND β′ SUBUNITS OF ESCHERICHIA COLI RNA POLYMERASE

The β and β′ subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are “split” into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.

RifR mutations in the beginning of the Escherichia coli rpoB gene.

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid β subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong RifR mutation lying outside the Rif region, which changed Val146 to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for RifR mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong RifR mutations were obtained when Val146 was mutated, and several weak RifR mutations were also isolated near position 146. The results define a new, N-terminal cluster of RifR mutations, in addition to the classical central Rif region.

STREPTOLYDIGIN-RESISTANT MUTANTS IN AN EVOLUTIONARILY CONSERVED REGION OF THE BETA ' SUBUNIT OF ESCHERICHIA COLI RNA POLYMERASE

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the β subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A.(1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the β‘ subunit. The mutation is the Phe793 → Ser substitution, which occurred in an evolutionarily conserved segment of the β‘ subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring α-amanitin resistance. Both streptolydigin and α-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.