A family of E. coli - P. ruminis shuttle-plasmids was constructed to allow the isolation
and characterization of gene promoters from the rumen bacterium P. ruminis. The
promoter rescue plasmid pBK was used to isolate a total of 4 genomic DNA fragments
that promoted transcription in P. ruminis strains 0/10. These promoters, and an
additional promoter, previously isolated from P. ruminis strain OR38 (Schoep, 1999),
were identified by their ability to initiate expression of a promoterless ermAM gene in
P. ruminis. Within 4 of the fragments, a total of 5 transcription start sites were identified
in P. ruminis using a novel, fluorescent-primer extension analysis protocol. Comparison
of promoters isolated in this and previous studies revealed a strong consensus RNA
polymerase DNA-binding motif, including the well characterized 35 and 10 elements.
Consensus sequences established for these elements were: TTgacA and AtAATAta
respectively, where bold upper-case font, regular upper-case, and lower-case fonts
represent conservation in 100%, 80%, and 70% of promoters respectively. The −10 and
−35 motifs were interspaced by 16 18 nt. Among the newly identified promoters, the
consensus for the 10 element was extended one nucleotide upstream and downstream
of the standard hexamer (boxed). These motifs were similar to those recognized by
eubacterial RNA polymerase containing the σ70-like factor. Promoters also contained
possible UP elements, and were significantly more curved than protein-coding regions.
Additional plasmid vectors were constructed, to allow the use of both the quantitative
SYBR green real time PCR and ß-glucuronidase assays, to examine 4 promoters in
depth. This showed a wide range of promoter strengths within the group. However, no
correlation was found between the composition and context of elements within P.
ruminis promoters, and promoter strength. A mutation within the 35 element of one
promoter revealed that promoter strength, and the choice of transcription start site were
both sensitive to single nucleotide
| Identifer | oai:union.ndltd.org:ADTP/221646 |
| Date | January 2004 |
| Creators | tdschoep@yahoo.com, Tobias Delavilla Schoep |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Tobias Delavilla Schoep |
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