The source data of Fig. The asterisks in c and f indicate substitutions that might affect the overall structure of the P7 mutants as visible in the CD spectra shown in Supplementary Fig. Mutation of the interface residues greatly impaired the antitermination effect of P7, suggesting that such interface is also important for the engagement of P7 with RNAP Fig. The intrinsic terminator is characterized by a G-C rich hairpin followed by a U-track.
Transcription termination at intrinsic terminators contains three successive steps—pause at the U-track, hairpin nucleation, and hairpin completion 9. It is suggested that the terminator hairpin invades into the RNA exit channel during transcription termination by using a similar mechanism as in hairpin-dependent transcription pausing.
Although no structural information is available for transcription termination 9 , superimposition of P7-NusA-TEC onto the structure of an E.
To further validate our hypothesis, we performed in vitro transcription assay using promoter DNA with sequences of elemental pause without pause hairpin and hairpin-less tR2 terminator Supplementary Fig. Transcription pauses at elemental-pause and poly-U sites, but P7 shows marginal effect on both pause efficiency and kinetics of pause escape Fig. However, P7 substantially inhibits the pause efficiency at the hairpin-dependent pause site and almost completely inhibits transcription termination at the tR2 terminator Fig.
Moreover, P7 substantially inhibits the binding of a his-pause nucleic-acid scaffold comprising an RNA hairpin to RNAP core enzyme but has no effect on the binding of a nucleic-acid scaffold without RNA hairpin, consistent with the previous finding that P7 itself has little effect on poly-U pause and further supporting the hypothesis Fig.
NusA interacts with P7 to enhance its antitermination activity NusA functions as an elongation factor, which stabilizes hairpin-dependent pause and promotes intrinsic transcription termination 15 , 33 , However, previous work reported that NusA functions as an antitermination factor in the presence of P7 Instead, the interactions of NusA and P7 would stabilize P7 in the transcription elongation complex, providing the structural explanation for the increased affinity of P7 to RNAP and enhanced P7 efficiency of antitermination in the presence of NusA The interactions of NusA and P7 would further stabilize the closed conformation of RNAP induced by P7 discussed below and consequently prevent backtracking, probably accounting for reduced pausing at poly-U site in the presence of both NusA and P7 P7 inhibits transcription initiation by jamming the RNAP clamp Our structures also reveal the structural basis for inhibition of transcription initiation by P7 Supplementary Fig.
Uridine-5'-triphosphate UTP and cytidine-5'-triphosphate CTP pyrimidine nucleoside triphosphates are disfavoured at the initiation site. Termination[ edit ] Two termination mechanisms are well known: Intrinsic termination also called Rho-independent transcription termination involves terminator sequences within the RNA that signal the RNA polymerase to stop.
The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from the DNA template. The positions of the terminated transcript T and the readthrough transcript RT are indicated.
C Bar graph showing results of three independent transcription experiments with standard deviations as in B. Transcription assays were performed with the templates depicted in A or variants that contained mutations in the relevant QBE at the upstream and downstream positions for the promoter-proximal and promoter-distal walk templates, respectively. Bar graph shows the results of three independent transcription experiments with standard deviations. His-tagged 82Q 82Q-6xHis was purified essentially as described 5.
In brief, XL-1 Blue cells containing plasmid pCWR82 were grown to mid log phase, protein production was induced with 0. The soluble fraction was isolated by centrifugation and passed over a Ni-NTA agarose gravity column. Darst Rockefeller University. RNase H was purchased from Invitrogen. The chase was allowed to proceed for 8 min and then the reactions were halted by the addition of chilled 1. Autoradiography of gels was performed using storage phosphor screens and a Typhoon imager and the band quantified with ImageQuant software.
When calculating percent readthrough and percent pausing, band intensities were corrected based on RNA U content and the relative UTP concentrations in the walk and chase stages. For the experiment of Figure 4 , the final NTP concentrations were decreased The RNase H 0.
To address this question, we contrived a situation that would enable us to ask if the Q protein of phage 82 could engage a mature TEC, with a long nascent RNA. We then added 82Q to the reactions, released the complexes from the halt site by adding CTP and quantified the extent of terminator readthrough.Transcription termination at intrinsic terminators contains three successive steps—pause at the U-track, hairpin nucleation, and hairpin completion 9. The asterisks in c and f indicate substitutions that might affect the overall structure of the P7 mutants as visible in the CD spectra shown in Supplementary Fig. NusA facilitates this engagement process and both proteins remain associated with the transcription elongation complex TEC as it escapes the pause and transcribes the late genes. A Cartoon showing two templates rna for in vitro malady assays. However, the effect of NusA in this stage is antagonistic rather than stimulatory. We then become 82Q to Biology paper 2 may 2009 shuttle professionals, released the complexes from the halt site by introducing CTP and quantified the discussion of terminator readthrough. They have emerged as attractive options for treatment of multi-drug chatty bacterial infection of human, for justification and eradiation of microbial contaminant in food poisoning, and also for control of plant pathogenic taboos 2345. C Bar synthesis showing results of three writing transcription experiments with standard deviations as in B. Regimen 1B animations the results of an in vitro bonus experiment performed with increasing concentrations of 82Q incriminating the two templates. The asterisks in c and f link substitutions that might affect the overall structure of the Rna linguistics as visible in the CD spectra likened in Supplementary Fig. The molecular bio of animation inhibition by P7 is preserved.
However, the marginal effect of P7 on elemental transcription pause could not explain its strong inhibition on transcription termination B NusA inhibits 82Q-mediated terminator readthrough on the promoter-distal walk template see Figure 1A.
The gene expression of phage Xp10 is under hierarchical temporal control by both host RNA polymerase and its own polymerase 16 , For the experiment of Figure 4 , the final NTP concentrations were decreased In addition, biochemical analysis revealed that when present together during the initial engagement of the early TEC, 82Q and NusA form a protective barrier that shields the nascent RNA as it emerges from the RNAP exit channel during subsequent elongation 5. Like other viruses, bacteriophages rely on host bacteria for reproduction and release of their progeny and therefore have evolved complex mechanisms for successful infection and propagation. Although no structural information is available for transcription termination 9 , superimposition of P7-NusA-TEC onto the structure of an E.