Experimental analysis of Helicobacter pylori transcriptional terminators suggests this microbe uses both intrinsic and factor-dependent termination.
In this study, we report experimental analysis of transcriptional terminators in the human pathogen Helicobacter pylori. Previous bioinformatics approaches came to differing conclusions regarding transcriptional termination in this bacterium. We used a reporter construct, the tnpR-encoded resolvase, to assess terminators. In our first experiments, we found that a subset of previously predicted intrinsic terminators for H. pylori are functional. In our second experiments, we used an unbiased screen to identify putative terminators and then characterized 18 of these. We found that these putative terminators overlap genomic regions that are either intergenic or intragenic. Using reverse transcription PCR, we showed that an intergenic terminator and an intragenic antisense terminator function at their endogenous loci. Additionally, we found that putative terminators contain features of both intrinsic and Rho-dependent termination, but that intrinsic terminators define the majority. We were unable to delete rho, however, in H. pylori, suggesting that it is essential and likely important. Finally, we carried out a mutational analysis of one of our randomly identified terminators that has both intrinsic and Rho-dependent features, and found that they are both functional. In conclusion, we found that H. pylori possesses numerous Rho-dependent and intrinsic terminators including some found in intragenic regions.
Castillo
Andrea R
AR
Department of Environmental Toxicology, University of California, Santa Cruz, CA, USA. castillo@etox.ucsc.edu
Arevalo
Sonia S
SS
Woodruff
Andrew J
AJ
Ottemann
Karen M
KM
D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D019295:Computational Biology; D016384:Consensus Sequence; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012234:Rho Factor / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D021901:DNA, Intergenic; D012329:RNA, Bacterial; D012234:Rho Factor