Selective promoter recognition by chlamydial sigma28 holoenzyme.
The sigma transcription factor confers the promoter recognition specificity of RNA polymerase (RNAP) in eubacteria. Chlamydia trachomatis has three known sigma factors, sigma(66), sigma(54), and sigma(28). We developed two methods to facilitate the characterization of promoter sequences recognized by C. trachomatis sigma(28) (sigma(28)(Ct)). One involved the arabinose-induced expression of plasmid-encoded sigma(28)(Ct) in a strain of Escherichia coli defective in the sigma(28) structural gene, fliA. The second was an analysis of transcription in vitro with a hybrid holoenzyme reconstituted with E. coli RNAP core and recombinant sigma(28)(Ct). These approaches were used to investigate the interactions of sigma(28)(Ct) with the sigma(28)(Ct)-dependent hctB promoter and selected E. coli sigma(28) (sigma(28)(Ec))-dependent promoters, in parallel, compared with the promoter recognition properties of sigma(28)(EC). Our results indicate that RNAP containing sigma(28)(Ct) has at least three characteristics: (i) it is capable of recognizing some but not all sigma(28)(EC)-dependent promoters; (ii) it can distinguish different promoter structures, preferentially activating promoters with upstream AT-rich sequences; and (iii) it possesses a greater flexibility than sigma(28)(EC) in recognizing variants with different spacing lengths separating the -35 and -10 elements of the core promoter.
Shen
Li
L
Department of Medicine, Boston Medical Center, Boston University School of Medicine, MA 02118, USA. lshen@bu.edu
Feng
Xiaogeng
X
Yuan
Yuan
Y
Luo
Xudong
X
Hatch
Thomas P
TP
Hughes
Kelly T
KT
Liu
Jun S
JS
Zhang
You-Xun
YX
D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012808:Sigma Factor / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C064971:FliA protein, Bacteria; D020035:Holoenzymes; D012808:Sigma Factor; D049452:Green Fluorescent Proteins; D012321:DNA-Directed RNA Polymerases