title: Mechanism of NusG-stimulated pausing, hairpin-dependent pause site selection and intrinsic termination at overlapping pause and termination sites in the Bacillus subtilis trp leader.
abstract: The Bacillus subtilis trpEDCFBA operon is regulated by TRAP-dependent transcription attenuation and translation repression mechanisms. Previous results showed that NusA and NusG cooperatively stimulate RNA polymerase pausing at U107 and U144 in the trp leader, and that NusG is required for pausing at U144 in vivo. Pausing at U107 and U144 participate in the attenuation and translation repression mechanisms, respectively, by providing additional time for TRAP binding. The intrinsic trp leader terminator overlaps the hairpin-dependent U144 pause site. Here, we conducted a systematic mutational analysis of the terminator/pause region. Deletion of the hairpin reduced pausing but did not affect pause site selection. Thus, hairpin-stimulated pausing is a more appropriate term than hairpin-dependent pausing for this pause site. In contrast, minor changes to the hairpin abolished termination. Sequences in the U-rich/T-rich tract following the hairpin affected termination and pausing differentially. The distance between the hairpin and the 3' end of the RNA dictates the position of termination, whereas the sequence downstream from the hairpin is responsible for pause site selection. NusA was found to increase both pausing and termination by reducing the rate of transcription. We also found that NusG-stimulated pausing is sequence specific and that NusG does not affect termination.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Yakhnin
authors.forename: Paul
authors.initials: P
authors.lastname: Babitzke
mesh_terms: D020121:5' Untranslated Regions; D001412:Bacillus subtilis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D001426:Bacterial Proteins; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial
references.citation: RNA. 2007 Nov;13(11):2020-33
references.pmid: 17881743
references.citation: Cell. 1996 Aug 9;86(3):485-93
references.pmid: 8756730
references.citation: Mol Microbiol. 2007 Oct;66(1):127-38
references.pmid: 17725561
references.citation: Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7090-5
references.pmid: 10860976
references.citation: Trends Microbiol. 2008 Mar;16(3):126-34
references.pmid: 18280161
references.citation: RNA. 2009 Jan;15(1):55-66
references.pmid: 19033375
references.citation: Cell. 1994 Jul 29;78(2):317-24
references.pmid: 8044843
references.citation: Mol Cell. 2002 Nov;10(5):1151-62
references.pmid: 12453422
references.citation: J Biol Chem. 1994 Jun 17;269(24):16597-604
references.pmid: 7515880
references.citation: Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):133-7
references.pmid: 7678334
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):8900-5
references.pmid: 19416863
references.citation: Science. 2001 Apr 27;292(5517):730-3
references.pmid: 11326100
references.citation: Nature. 1999 Sep 16;401(6750):235-42
references.pmid: 10499579
references.citation: Mol Cell. 2004 Apr 9;14(1):117-26
references.pmid: 15068808
references.citation: Mol Cell. 2007 Dec 28;28(6):991-1001
references.pmid: 18158897
references.citation: J Biol Chem. 2000 Feb 11;275(6):4519-24
references.pmid: 10660627
references.citation: Nature. 2007 Jul 12;448(7150):157-62
references.pmid: 17581590
references.citation: Cell. 2001 Nov 16;107(4):437-49
references.pmid: 11719185
references.citation: Mol Cell. 1999 Apr;3(4):495-504
references.pmid: 10230402
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: Mol Cell. 2006 Nov 17;24(4):547-57
references.pmid: 17114058
references.citation: J Mol Biol. 1995 Dec 15;254(5):808-14
references.pmid: 7500352
references.citation: Annu Rev Genet. 2005;39:47-68
references.pmid: 16285852
references.citation: J Biol Chem. 1990 Sep 5;265(25):15145-53
references.pmid: 1697586
references.citation: Methods Enzymol. 1996;274:334-53
references.pmid: 8902817
references.citation: J Mol Biol. 1997 Apr 25;268(1):54-68
references.pmid: 9149141
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 21;105(42):16131-6
references.pmid: 18852477
references.citation: J Bacteriol. 2000 Apr;182(7):1819-27
references.pmid: 10714985
references.citation: J Mol Biol. 1993 Sep 5;233(1):25-42
references.pmid: 8377190
references.citation: Cell. 2002 Apr 19;109(2):193-203
references.pmid: 12007406
references.citation: Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8793-7
references.pmid: 7568019
references.citation: J Mol Biol. 1997 Aug 1;270(5):696-710
references.pmid: 9245598
references.citation: Nature. 1995 Apr 20;374(6524):693-700
references.pmid: 7715723
references.citation: J Biol Chem. 1998 Aug 7;273(32):20494-503
references.pmid: 9685405
references.citation: Mol Cell. 2007 Apr 13;26(1):117-29
references.pmid: 17434131
references.citation: Mol Cell. 2007 Aug 3;27(3):406-19
references.pmid: 17679091
references.citation: J Bacteriol. 1995 Nov;177(22):6362-70
references.pmid: 7592410
references.citation: Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11067-72
references.pmid: 12161562
references.citation: Mol Microbiol. 1998 Jun;28(6):1187-97
references.pmid: 9680208
references.citation: J Mol Biol. 2003 Nov 21;334(2):205-13
references.pmid: 14607113