Konstantin Severinov

Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription

Expressed protein ligation is a novel protein semi-synthesis method that permits the in vitro ligation of a chemically synthesized C-terminal segment of a protein to a recombinant N-terminal segment fused through its C terminus to an intein protein splicing element. In principle, the practical convenience of this method, combined with the expanded opportunities in protein engineering that it provides, makes it well suited for probing the molecular basis of complex processes such as transcription. Here we describe the successful application of expressed protein ligation to the ∼600 amino acid ς70 subunit of Escherichia coli RNA polymerase. The resulting semi-synthetic ς70 constructs are shown to be fully functional and have been used to map the binding region of the bacteriophage T4 anti-sigma protein, AsiA, to within amino acids 567–600 of ς70. The success of these semi-synthesis studies sets the stage for the future generation of semi-synthetic ς70 molecules in which unnatural amino acids and biophysical probes are site-specifically incorporated in the RNA polymerase complex.

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.

Role of Escherichia coli RNA polymerase alpha subunit in modulation of pausing, termination and anti-termination by the transcription elongation factor NusA.

The alpha subunit (alpha) of RNA polymerase (RNAP) is critical for assembly of polymerase and positive control of transcription initiation in Escherichia coli. Here, we report that alpha also plays a role in transcription elongation, and this involves a direct interaction between alpha and NusA factor. During in vitro transcription without NusA, alpha interacts with the nascent RNA, as revealed by photocrosslinking. When NusA is present, RNA crosslinks to NusA rather than to alpha. We show that this NusA‐RNA interaction is diminished during transcription with an RNAP mutant that lacks the C‐terminus of alpha beyond amino acid 235, including the so‐called alpha CTD. The absence of alpha CTD also affects NusAs ability to enhance transcription pausing, termination at intrinsic terminators and anti‐termination by the phage lambda Q anti‐terminator, but not anti‐termination by the lambda N anti‐terminator. NusA functions are not recovered even when transcription with mutant RNAP is done with excess NusA, a condition which does restore NusA‐RNA crosslinking. By affinity chromatography, we show that NusA interacts directly with alpha, and also with beta and beta’, but not with mutant alpha. Hence, alpha‐NusA interaction is vital for the control of transcript elongation and termination.

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the β and β′-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always precedingrpoC. Here, we show that in Escherichia colithe two genes can be fused and that the resulting 2751-amino acid β::β′ fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro. The results establish that the C terminus of the β subunit and the N terminus of the β′ subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivomay occur co-translationally.

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.

Disruption of Escherichia coli HepA, an RNA Polymerase-associated Protein, Causes UV Sensitivity*

During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of thehepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein. We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity ς70 subunit with a 1:1 stoichiometry and a dissociation constant (K d ) of 75 nm. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.

Pribnow Box Recognition and Melting by Escherichia coli RNA Polymerase

The core RNA polymerases from bacterial and eukaryotic cells, which are homologous in structure and function (Allison et al. 1985; Biggs et al. 1985; Ahearn et al. 1987; Sweetser et al. 1987; Darst et al. 1989, 1991; Schultz et al. 1993; Polyakov et al. 1995), are catalytically active in RNA chain elongation but are incapable of promoter recognition and specific initiation. Promoter-specific transcription initiation requires additional protein factors. In bacteria, specific initiation by RNA polymerase (RNAP) requires a single polypeptide known as a σ factor, which binds to core RNAP to form the holoenzyme (Burgess et al. 1969; Travers and Burgess 1969). One primary σ factor directs the bulk of transcription during exponential growth. Specialized, alternative σ factors direct transcription of specific regulons during unusual physiological or developmental conditions (reviewed in Helmann and Chamberlin 1988; Gross et al. 1992). The primary and most of the alternative σ factors comprise a highly homologous family of proteins (Stragier et al. 1985; Gribskov and Burgess 1986) with four regions of highly conserved amino acid sequence (Fig. 1; reviewed in Lonetto et al. 1992). Based on the results of genetic and biochemical experiments, specific functions have been assigned to some of the conserved regions(summarized in Fig. 1).

Chapter 8:Regulation of RNA Polymerase through its Active Center

RNA polymerase (RNAP) is arguably the most highly regulated enzyme in the cell. The ability of RNAP to sense and integrate signals from various factors, including nucleic acid sequences, protein regulators, and small molecules largely determines the ability of the cell, and ultimately the entire org...