HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA. Instead, a genetic screen reveals that the overexpression of glutamyl-tRNA synthetase (GltX) suppresses the toxicity of HipA. We show that HipA phosphorylates conserved Ser(239) near the active center of GltX and inhibits aminoacylation, a unique example of an aminoacyl-tRNA synthetase being inhibited by a toxin encoded by a toxin-antitoxin locus. HipA only phosphorylates tRNA(Glu)-bound GltX, which is consistent with the earlier finding that the regulatory motif containing Ser(239) changes configuration upon tRNA binding. These results indicate that HipA mediates persistence by the generation of "hungry" codons at the ribosomal A site that trigger the synthesis of (p)ppGpp, a hypothesis that we verify experimentally.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP b subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

BackgroundTranscription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.ResultsHere we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH.ConclusionsWe demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

Fidelity of template-dependent nucleic acid synthesis is the main determinant of stable heredity and error-free gene expression. The mechanism (or mechanisms) ensuring fidelity of transcription by DNA-dependent RNA polymerases (RNAPs) is not fully understood. Here, we show that the 3' end-proximal nucleotide of the nascent transcript stimulates hydrolysis of the penultimate phosphodiester bond by providing active groups and coordination bonds to the RNAP active center. This stimulation is much higher in the case of misincorporated nucleotide. We show that during transcription elongation, the hydrolytic reaction stimulated by misincorporated nucleotides proofreads most of the misincorporation events and thus serves as an intrinsic mechanism of transcription fidelity.

Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities.

Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III -synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, while not causing termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multi-subunit RNA polymerases.Termination of transcription is an obligatory step following synthesis of the transcript, which leads to dissociation of RNA polymerase (RNAP) and the transcript from the template DNA. However, apparently different mechanisms are utilized by evolutionary conserved multi-subunit RNAPs from bacteria, archaea, and three eukaryotic RNAPs to terminate transcription (1-3). Pol III terminates after synthesis of a poly-U stretch (4, 5), and most studies have focused on the efficiency of recognition of the poly-T (on the nontemplate strand) termination signal (6). Both upstream and downstream sequences were shown to influence efficiency of recognition (7). However, the events leading to termination on the poly-T signal, i.e. dissociation of Pol III from the template, are not known.We investigated this problem by using assembled elongation complexes, a technique successfully used to investigate various RNAPs (8-11). These complexes, assembled with purified RNAP, synthetic complementary template and non-template DNA strands and RNA, allow skipping the step of initiation and, therefore, excluding any accessory factors from the reaction. Complexes were immobilized on streptavidin beads via biotin on the 5′ end of the non-template strand (scheme in Fig. 1A). The RNA in complexes was radioactively labeled by incorporation of radioactive NMP (12). First, we analyzed transcription through poly-T signals of various lengths by purified S. cerevisiae Pol III. As seen from Fig. 1A, at poly-T signals longer than 5 nucleotides, transcripts finishing at the end the poly-T signal were formed. On long poly-T signals (12T), transcription was stopping predominantly after 6 th -10 th T (T 12 template in Fig. 1B, lane 10). No stopping was observed on homopolymeric tracts other than poly-T (Fig. S1). We tested, if transcripts ending with a poly-U stretch were released from the template as a result of termination. This can be done by analysis of transcripts in the supernatant and immobilized fractions of the reaction ("super" and "beads" fractions, respectively, in scheme of Fig. 1B). As seen from Fig. 1B, while RNAs resulting from transcription to the end of tem...

21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.