Promoter prediction in the rhizobia
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abstract
The ability to recognize and predict non-σ54promoters in the alphaproteobacteria is not well developed. In this study, 25 experimentally verifiedSinorhizobium melilotipromoter sequences were compiled and used to predict the location of other related promoters in theS. melilotigenome. Fourteen candidate predictions were targeted for verification and of these at least 12 proved to be genuine promoters. As a result, the experimental identification of 12 novel promoters linked to genesrpoD,topA,rpmJ,trpS,ropB1,metC,rpsT,secE,trkHand three tRNA genes is reported. In all, 99 predicted and verified promoters are reported, including those linked with 13 tRNA genes, eight ribosomal protein genes and a number of other physiologically important or essential genes. On the basis of sequence conservation and a mutational analysis of promoter activity, the −35 and −10 consensus for these promoters is 5-CTTGAC-N17-CTATAT. This promoter structure, which seems to be widely conserved amongst several other genera in the alphaproteobacteria, shares significant similarity with, but is skewed by a 1 nt step from, the canonicalEscherichia coliσ70promoter. Perhaps this difference is responsible for the observation thatS. melilotipromoters are often poorly expressed inE. coli. In this regard, expression data from plasmid-bornegfp-reporter fusions to eight of theS. melilotipromoters verified in this work revealed that while these promoters were very active inS. melilotiandAgrobacterium tumefaciensonly very low, near-background activity was detected inE. coli.
