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.

Microcin J25 (MccJ25) is a 21-amino acid peptide inhibitor active against the DNA-dependent RNA polymerase of Gram negative bacteria. Previously, the structure of MccJ25 was reported to be a head-to-tail circle, cyclo(-G(1)GAGHVPEYF(10)VGIGTPISFY(20)G-). On the basis of biochemical studies, mass spectrometry, and NMR, we show that this structure is incorrect, and that the peptide has an extraordinary structural fold. MccJ25 contains an internal lactam linkage between the alpha-amino group of Gly1 and the gamma-carboxyl of Glu8. The tail (Tyr9-Gly21) passes through the ring (Gly1-Glu8), with Phe19 and Tyr20 straddling each side of the ring, sterically trapping the tail in a noncovalent interaction we call a lassoed tail.

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...

The new mutations affect ␤ amino acids in evolutionarily conserved segments G, G, and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center. We also report that previously known rpoB (RNAP ␤ subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25. We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

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.

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two Mg 2þ mechanism and is assisted by the 3′ end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (β′ His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3′ end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the β′ His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria. C atalysis by the active center of RNA polymerase (RNAP) is thought to proceed through a general two Mg 2þ ion mechanism (1-4). Structural and biochemical studies, however, revealed that during phosphodiester bond synthesis a flexible domain of the active center, the trigger loop (TL), is also required for catalysis (5-10). The TL was proposed to act by orienting the triphosphate moiety of the incoming NTP for efficient attack by the 3′ hydroxyl of RNA (5-7, 10). In crystal structures of RNAP elongation complexes, the TL was observed in two distinct states, folded and unfolded. Only in the folded conformation are amino acids of the TL close enough to the catalytic center to be able to participate in the reaction, so the folded form of the TL is assumed to be the catalytically active state (6-9).The active center of RNAP is also able to perform hydrolysis of the phosphodiester bond of the transcript (3,11,12). This reaction almost exclusively proceeds at the penultimate (second) phosphodiester bond at the 3′ end of RNA transcript (12, 13). Such preference is because of the fact that the rate of hydrolysis of the second phosphodiester bond is hugely increased by assistance from the 3′ end NMP of the RNA (transcript-assisted hydrolysis) (12). Transcript-assisted hydrolysis proceeds in a 1-bp backtracked conformation of the elongation complex, in which the 3′ end NMP of the transcript disengages from the complementary template base to assist with the second phosphodiester bond hydrolysis (12,14). The 3′ end NMP was proposed to provide its chemical groups to the active center of RNAP to increase the affinity to the second Mg 2þ ion (Mg 2þ II) and/or to position the attacking water molecule (12). Reaction is especially efficient when RNAP incorporates an erroneous nucleotide, given that the misincorporated elongation complex is stabilized in the 1-bp backtracked state. Hydrolysis of the second phosphodiester bond, therefore, is thought to be important for proofreading of transcript...

Bacterial RNA polymerase is able to initiate transcription with adenosine-containing cofactor NAD+, which was proposed to result in a portion of cellular RNAs being ‘capped’ at the 5′ end with NAD+, reminiscent of eukaryotic cap. Here we show that, apart from NAD+, another adenosine-containing cofactor FAD and highly abundant uridine-containing cell wall precursors, UDP-Glucose and UDP-N-acetylglucosamine are efficiently used to initiate transcription in vitro. We show that the affinity to NAD+ and UDP-containing factors during initiation is much lower than their cellular concentrations, and that initiation with them stimulates promoter escape. Efficiency of initiation with NAD+, but not with UDP-containing factors, is affected by amino acids of the Rifampicin-binding pocket, suggesting altered RNA capping in Rifampicin-resistant strains. However, relative affinity to NAD+ does not depend on the −1 base of the template strand, as was suggested earlier. We show that incorporation of mature cell wall precursor, UDP-MurNAc-pentapeptide, is inhibited by region 3.2 of σ subunit, possibly preventing targeting of RNA to the membrane. Overall, our in vitro results propose a wide repertoire of potential bacterial RNA capping molecules, and provide mechanistic insights into their incorporation.