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Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro.

ABSTRACT: We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential -10 and -35 elements, analogous to Escherichia coli promoters recognized by E-sigma70. We identified a novel AT-rich region immediately downstream of the -35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the -35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

SUBMITTER: Tan M

PROVIDER: S-EPMC107176 | biostudies-literature | 1998 May

REPOSITORIES: biostudies-literature

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Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro.

Tan M M Gaal T T Gourse R L RL Engel J N JN

Journal of bacteriology 19980501 9

We have characterized the Chlamydia trachomatis ribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purified C. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential -10 and -35 elements, analogous to Escherichia coli promoters recognized by E-sigma70. We identified a novel AT-rich region immediately downstream of the -35 region. The effect of this region was specific for C. trachomatis RNA polym ...[more]

PMID: 9573186

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Project description:Introduction Chlamydia trachomatis (C. trachomatis) is a Gram-negative bacterium and a common human pathogen. The World Health Organization (WHO) estimates that over 130 million people are infected with C. trachomatis globally each year and with increasing incidence. C. trachomatis causes long-lasting and recurrent infections that over time induce severe tissue damage in the female genital tract that can lead to ectopic pregnancy and infertility. Thus, the human immune system fails to control and eradicate C. trachomatis during primary infection and fails to develop protective immunity against secondary infections. In vivo infection models, using complement knock out mice, suggest that the complement system is critically involved in both anti-chlamydial immunity and infection-induced pathology. To increase our understanding of complement-mediated immunity against C. trachomatis we analyzed global complement deposition on serum-incubated C. trachomatis by mass spectrometry. Methods Purified C. trachomatis was incubated in seronegative normal human serum (NHS) or heat-inactivated normal human serum (HI-NHS) for 30 min, thoroughly washed, and processed for mass spectrometry. All samples were lysed, reduced and alkylated and digested with trypsin. Some samples were chemically modified to acetylate free amino groups (N-terminal and lysine amino groups) before trypsin digestion. Peptides were analyzed on a UltimateTM 3500 RSLCnano coupled to a Q Exactive HF-X mass spectrometer. Raw data files were searched against the Uniprot human reference proteome using MaxQuant. Results We demonstrate that C. trachomatis elicits potent complement activation demonstrated by deposition of both early and late complement factors together with several complement regulators. We further demonstrate proteolytically processing of complement C3b to “inactive” C3 cleavage fragments. Conclusion We demonstrate the deposition of several novel complement-associated proteins and -cleavage fragments on the surface of C. trachomatis.

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Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro.

Publications

Mutational analysis of the Chlamydia trachomatis rRNA P1 promoter defines four regions important for transcription in vitro.

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