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Tina M Henkin – 1st expert on this subject based on the ideXlab platform

  • non conserved residues in clostridium acetobutylicum trnaala contribute to trna tuning for efficient Antitermination of the alas t box riboswitch
    Life, 2015
    Co-Authors: Frank J Grundy, Tina M Henkin


    The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient Antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs Antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal Antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient Antitermination.

  • kinetic analysis of trna directed transcription Antitermination of the bacillus subtilis glyqs gene in vitro
    Journal of Bacteriology, 2004
    Co-Authors: Frank J Grundy, Tina M Henkin


    Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription Antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent Antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and Antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on Antitermination efficiency was analyzed. We found that addition of tRNAGly can promote Antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

  • trna requirements for glyqs Antitermination a new twist on trna
    RNA, 2003
    Co-Authors: Mary R Yousef, Frank J Grundy, Tina M Henkin


    Transcription Antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNAGly. Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNAGly were generated to identify additional tRNA elements required for Antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3′ end of the tRNA blocked Antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of 1 nucleotide between the acceptor stem and the 3′ UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited Antitermination function, as was previously demonstrated for tRNATyr-directed Antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished Antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3′ end of the tRNA for interaction with the antiterminator bulge and Antitermination.

David I Friedman – 2nd expert on this subject based on the ideXlab platform

  • evidence that the promoter can influence assembly of Antitermination complexes at downstream rna sites
    Journal of Bacteriology, 2006
    Co-Authors: Ying Zhou, Eric R. Olson, Mark A Mozola, Karla S Henthorn, Susan Brown, Gary N Gussin, David I Friedman


    The N protein of phage λ acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated Antitermination. We present evidence that the associated promoter, in this case the λ early PR promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in PR, at positions −14 and −45 (yielding PR-GA), reduces effectiveness of N modification, while an additional mutation at position −30 (yielding PR-GCA) suppresses this effect. In vivo, the level of PR-GA-directed transcription was twice as great as the wild-type level, while transcription directed by PR-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at PR-GA in vitro was roughly one-third the rate for wild-type PR. We ascribe this apparent discrepancy to an effect of the mutations in PR-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active Antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking Antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

  • analyzing transcription Antitermination in lambdoid phages encoding toxin genes
    Methods in Enzymology, 2003
    Co-Authors: Melody N Neely, David I Friedman


    Publisher Summary This chapter analyzes the transcription of Antitermination in lambdoid phages encoding toxin genes. Lambdoid phages carry the genes encoding Shiga toxin (Stx A and B): most commonly, the genes encoding the two subunits of either the stx1 or stx2 genes. The two best-studied stx-carrying phages are Stx1 phage H-19B and Stx2 phage 933W. Four methods of analysis were used to determine whether H-19B has an Antitermination system similar to that of λ and assess its specific mechanism of action: DNA sequence determination of regions of the H-19B genome that may play a role in Antitermination based on studies of λ N-mediated Antitermination; growth of H-19B in E. coli mutants defective for λ Antitermination; construction and analysis of H-19B phage mutants; and reporter constructs designed to study Antitermination mediated by the H-19B N protein. Analysis of H-19B Q-mediated Antitermination involved similar methods as those employed in analysis of N-mediated Antitermination: sequence determination of the late regulatory region of H-19B, which includes the Q gene, the Prʹ promoter with its associated qut site, and the t R ʹ terminator, as well as stx and lysis genes; phage mutants; and reporter–terminator constructs to measure factors required for Antitermination at downstream terminators.

  • requirement for nusg for transcription Antitermination in vivo by the λ n protein
    Journal of Bacteriology, 2002
    Co-Authors: Ying Zhou, Donald L Court, Joshua J Filter, Max E Gottesman, David I Friedman


    Transcription Antitermination by the bacteriophage λ N protein is stimulated in vitro by the Escherichia coli NusG protein. Earlier work suggested that NusG was not required for N activity in vivo. Here we present evidence that NusG also stimulates N-mediated transcription Antitermination in intact cells.

Donald L Court – 3rd expert on this subject based on the ideXlab platform

  • structural basis for rna recognition by nusb and nuse in the initiation of transcription Antitermination
    Nucleic Acids Research, 2011
    Co-Authors: Jason Stagno, Donald L Court, Amanda S Altieri, Mikhail Bubunenko, Sergey G Tarasov, Jess Li, Andrew R Byrd, Xinhua Ji


    Processive transcription Antitermination requires the assembly of the complete Antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda Antitermination site. Combining these data with known interactions amongst Antitermination factors suggests a specific model for the assembly of the complete Antitermination complex.

  • structural and functional analysis of the e coli nusb s10 transcription Antitermination complex
    Molecular Cell, 2009
    Co-Authors: Hehsuan Hsiao, Donald L Court, Mikhail Bubunenko, Max E Gottesman, Markus C Wahl, G Weber, Henning Urlaub


    Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription Antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for Antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA Antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive Antitermination.

  • essentiality of ribosomal and transcription Antitermination proteins analyzed by systematic gene replacement in escherichia coli
    Journal of Bacteriology, 2007
    Co-Authors: Mikhail Bubunenko, Teresa Baker, Donald L Court


    We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA Antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription Antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.