Difference between revisions of "Part:BBa I20530"

Revision as of 23:30, 17 June 2011

Consensus promoter for phage K1-5 RNA polymerase

Useful for orthogonal transcription with cognate K1-5 RNA polymerase. Cognate RNA pol encoded by BBa_I20531.

From Scholl et al. Journal of Molecular Biology, Volume 335, Issue 5, 30 January 2004, Pages 1151-1171

"Most SP6 and K1-5 transcription occurs using their respective phage-encoded RNAPs. Several SP6 promoters have been identified and cloned and transcriptional activity with respect to promoter sequence has been examined comprehensively by saturation mutagenesis.[33.] and [34.] On the basis of this information we have identified 12 phage RNAP promoters in the SP6 genome. Only the promoter upstream of ORF31 conforms exactly to the consensus. Three promoter-like sequences that contain one or more nucleotide changes at conserved positions, including+1G that is required for SP6 RNAP transcription initiation in vitro, were found but are likely not to be functional."

"The consensus promoter sequences for SP6 and K1-5 differ at positions −10, −11, −12 (Table 2 and Table 3). The SP6 consensus −12G and −10T are not major determinants of promoter activity in vivo or in vitro but any substitution at −11G is inactivating.34 All K1-5 promoters have a pyrimidine at −11, suggesting that SP6 RNAP will not transcribe K1-5 DNA and vice versa. A comparable specificity of the phage RNAP for its cognate promoters is seen throughout the T7 group; for example, T7 RNAP does not recognize T3 promoters and vice versa. However, a single amino acid change in T7 RNAP is sufficient to switch its specificity to T3 promoters, and a change at −11 in a T7 promoter is sufficient to allow its recognition by T3 RNAP.[36.] and [37.] Two changes at positions −9 and −8 of the SP6 promoter to those of a T7 promoter allow recognition by T7 RNAP in vitro.38 All SP6 and most K1-5 promoters have the sequence 5′-GA at these positions, suggesting that the K1-5 genome cannot be transcribed efficiently by T7 RNAP."

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Acknowledgement

[http://www.biosci.utexas.edu/mgm/People/Faculty/profiles/?id=1707 Prof. Ian J. Molineux] of UT Austin suggested the K1-5 RNA polymerase system as an orthogonal transcription system distinct from T3, T7, SP6, et al.

Sequence and Features