title: Mutagenesis of the P2 promoter of the major outer membrane protein gene of Chlamydia trachomatis.
abstract: On the basis of position from the transcription start site, the P2 promoter of the gene encoding the major outer membrane protein (ompA) of Chlamydia trachomatis consists of a -35 hexamer region of -42 aaaaaga TATACAaa -28 and an unusual, GC-rich -10 hexamer region of -13 tTATCGCt -6. The P2 promoter was analyzed by in vitro transcription of templates containing deletions and site-specific mutations. The 5' extent of P2 was located at bp -42. Replacement of wild-type sequence with two G's at positions -41 and 40, -35 and 34, and -29 and 28 resulted in severely decreased transcription. Additionally, the spacing between the -35 and -10 hexamers could not be shortened without adversely affecting in vitro activity. Substitution of G at position -13, -10, -7, or -6 had little or no effect on transcription, whereas substitution of G at -11 or -12 significantly decreased promoter strength. Triple point mutations which changed the -10 hexamer from TATCGC to TATTAT,TATATT, or TATAAT had little effect on promoter activity. Unlike the partially purified C. trachomatis sigma66-RNA polymerase used in this study, purified Escherichia coli sigma70-RNA polymerase did not recognize the wild-type P2 promoter. Mutant P2 templates with -10 hexamers that resembled the consensus recognition site were transcribed by E. coli holoenzyme in vitro, suggesting that C. trachomatis sigma66-RNA polymerase has special promoter recognition properties not found in E. coli sigma70-holoenzyme.
authors.affiliation: Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Douglas
authors.forename: T P
authors.initials: TP
authors.lastname: Hatch
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002474:Cell-Free System; D002692:Chlamydia trachomatis / Q000235:genetics*; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012808:Sigma Factor / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D012808:Sigma Factor; C096461:sigma 66 factor, Chlamydia; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases