Length of guanosine homopolymeric repeats modulates promoter activity of subfamily II tpr genes of Treponema pallidum ssp. pallidum.
In Treponema pallidum, homopolymeric guanosine repeats of varying length are present upstream of both Subfamily I (tprC, D, F and I) and II (tprE, G and J) tpr genes, a group of potential virulence factors, immediately upstream of the +1 nucleotide. To investigate the influence of these poly-G sequences on promoter activity, tprE, G, J, F and I promoter regions containing homopolymeric tracts with different numbers of Gs, the ribosomal binding site and start codon were cloned in frame with the green fluorescent protein reporter gene (GFP), and promoter activity was measured both as fluorescence emission from Escherichia coli cultures transformed with the different plasmid constructs and using quantitative RT-PCR. For tprJ, G and E-derived clones, fluorescence was significantly higher with constructs containing eight Gs or fewer, while plasmids containing the same promoters with none or more Gs gave modest or no signal above the background. In contrast, tprF/I-derived clones induced similar levels of fluorescence regardless of the number of Gs within the promoter. GFP mRNA quantification showed that all of the promoters induced measurable transcription of the GFP gene; however, only for Subfamily II promoters was message synthesis inversely correlated to the number of Gs in the construct.
Department of Medicine, University of Washington, Seattle, WA, USA.
Lorenzo
L
Giacani
Sheila
S
Lukehart
Arturo
A
Centurion-Lara
D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014210:Treponema pallidum / Q000235:genetics* / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; C406409:Tpr protein, Treponema; D049452:Green Fluorescent Proteins