No abstract

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2␣, ␤, ␤, and . However, in many Grampositive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called ␦. For over three decades since its identification, it had been thought that ␦ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that ␦ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that ␦ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of ␦. Performing biochemical and mutational analysis, we show that Bacillus subtilis ␦ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, ␦ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of ␦. Using transcription assay, we show that ␦-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that ␦ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the ؊35 promoter element for transcription activation suggests that ␦ functions as a transcriptional regulator.Transcription is the first step in gene regulation in bacteria in which RNA polymerase (RNAP) 3 together with different factors and transcriptional regulators control the gene expression. Bacterial RNAP core enzyme contains five conserved subunits: 2 ␣, ␤, ␤Ј, and . A specificity factor associates with RNAP core enzyme to form RNAP holo enzyme that is able to recognize and initiate transcription at promoters.In certain Gram-positive bacteria, including Bacillus subtilis and Staphylococcus aureus, an additional factor, called ␦, is associated with RNAP. The ␦ factor was first identified in 1975 during the purification of RNAP from phage (SP01)-infected B. subtilis (1). The protein copurified with RNAP, and therefore it was thought that ␦ functions as a subunit of RNAP. Attempts were made to characterize the functional role of the protein in transcription. Several reports suggested that ␦ was involved in promoter selection (2-5) and functioned together with A as an initiation subunit of RNAP (6, 7) or as an allosteric modulator of RNAP conformation in both initiation and the RNAP core recycling phase (5). Other reports showed that ␦ and A bind to RNAP core with negative cooperativity (8, 9), and ␦ has no effect on transcription initiation, the rate of elongation, or termination (5). Using in vitro transcription assays, several groups showed that ␦ enhances the production of transcripts from certain promoters. This increase in transcript yield in the presence of ␦ is attributed to the recycling of RNAP possibly by ␦-mediated release of RNAP from the elongation complex following transcription termination or by inhibiting the formation of s...

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation.

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.