Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation.

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription.

The TFIIE-related Rpc82 subunit of RNA polymerase III interacts with the TFIIB-related transcription factor Brf1 and the polymerase cleft for transcription initiation.

Rpc82 is a TFIIE-related subunit of the eukaryotic RNA polymerase III (pol III) complex. Rpc82 contains four winged-helix (WH) domains and a C-terminal coiled-coil domain. Structural resolution of the pol III complex indicated that Rpc82 anchors on the clamp domain of the pol III cleft to interact with the duplex DNA downstream of the transcription bubble. However, whether Rpc82 interacts with a transcription factor is still not known. Here, we report that a structurally disordered insertion in the third WH domain of Rpc82 is important for cell growth and in vitro transcription activity. Site-specific photo-crosslinking analysis indicated that the WH3 insertion interacts with the TFIIB-related transcription factor Brf1 within the pre-initiation complex (PIC). Moreover, crosslinking and hydroxyl radical probing analyses revealed Rpc82 interactions with the upstream DNA and the protrusion and wall domains of the pol III cleft. Our genetic and biochemical analyses thus provide new molecular insights into the function of Rpc82 in pol III transcription.

Genetic regulation of the yefM-yoeB toxin-antitoxin locus of Streptococcus pneumoniae.

Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeB(Spn) operon studied here. Through footprinting analyses, we showed that purified YefM(Spn) antitoxin and the YefM-YoeB(Spn) TA protein complex bind to a palindrome sequence encompassing the -35 region of the main promoter (P(yefM2)) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P(yefM2), since YefM(Spn) behaved as a weak repressor with YoeB(Spn) as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P(yefM2). BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB(Spn) locus, the boxAC element provided an additional weak promoter, P(yefM1), upstream of P(yefM2) which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P(yefM1) was constitutive albeit weaker than P(yefM2). Intriguingly, the coupling of the boxAC element to P(yefM1) and yefM(Spn) in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeB(Spn) locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB(Spn) locus suggested that its presence may provide a selective advantage to the bacterium.

Mapping the protein interaction network for TFIIB-related factor Brf1 in the RNA polymerase III preinitiation complex.

TFIIB-related factor Brf1 is essential for RNA polymerase (Pol) III recruitment and open-promoter formation in transcription initiation. We site specifically incorporated a nonnatural amino acid cross-linker into Brf1 to map its protein interaction targets in the preinitiation complex (PIC). Our cross-linking analysis in the N-terminal domain of Brf1 indicated a pattern of multiple protein interactions reminiscent of TFIIB in the Pol active-site cleft. In addition to the TFIIB-like protein interactions, the Brf1 cyclin repeat subdomain is in contact with the Pol III-specific C34 subunit. With site-directed hydroxyl radical probing, we further revealed the binding between Brf1 cyclin repeats and the highly conserved region connecting C34 winged-helix domains 2 and 3. In contrast to the N-terminal domain of Brf1, the C-terminal domain contains extensive binding sites for TBP and Bdp1 to hold together the TFIIIB complex on the promoter. Overall, the domain architecture of the PIC derived from our cross-linking data explains how individual structural subdomains of Brf1 integrate the protein network from the Pol III active center to the promoter for transcription initiation.

Molecular and structural characterization of the PezAT chromosomal toxin-antitoxin system of the human pathogen Streptococcus pneumoniae.

The chromosomal pezT gene of the Gram-positive pathogen Streptococcus pneumoniae encodes a protein that is homologous to the zeta toxin of the Streptococcus pyogenes plasmid pSM19035-encoded epsilon-zeta toxin-antitoxin system. Overexpression of pezT in Escherichia coli led to severe growth inhibition from which the bacteria recovered approximately 3 h after induction of expression. The toxicity of PezT was counteracted by PezA, which is encoded immediately upstream of pezT and shares weak sequence similarities in the C-terminal region with the epsilon antitoxin. The pezAT genes form a bicistronic operon that is co-transcribed from a sigma(70)-like promoter upstream of pezA and is negatively autoregulated with PezA functioning as a transcriptional repressor and PezT as a co-repressor. Both PezA and the non-toxic PezA(2)PezT(2) protein complex bind to a palindrome sequence that overlaps the promoter. This differs from the epsilon-zeta system in which epsilon functions solely as the antitoxin and transcriptional regulation is carried out by another protein designated omega. Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site. In the PezA(2)PezT(2) complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance.

The yefM-yoeB toxin-antitoxin systems of Escherichia coli and Streptococcus pneumoniae: functional and structural correlation.

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM(Spn)) and toxin (YoeB(Spn)) products. We showed that overproduction of YoeB(Spn) is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB(Spn)-mediated toxicity could be reversed by the cognate antitoxin, YefM(Spn), but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.