Thu, 21 Aug 2008 01:08:17 BST CiteULike: msuarezdiez's Xu http://www.citeulike.org/user/msuarezdiez/author/Xu CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/msuarezdiez/article/2860863 <i>Journal of Molecular Biology (September 1999), pp. 19-37.</i><br /><br />The in vivo activities of seven constitutive promoters in Escherichia coli have been determined as functions of growth rate in wild-type relA+spoT+strains with normal levels of guanosine tetraphosphate (ppGpp) and in ppGpp-deficient relAspoT derivatives. The promoters include (i) the spc ribosomal protein operon promotor Pspc; (ii) the -lactamase gene promotor Pblaof plasmid pBR322; (iii) the PLpromoter of phage ; (iv) and (v) the replication control promoters PRNAIand PRNAIIof plasmid pBR322; and (vi) and (vii) the P1 and P2 promoters of the rrnB ribosomal RNA operon. Each strain carried an operon fusion consisting of one of the respective promoter regions linked to lacZ and recombined into the chromosome at the mal locus of alac deletion strain. The amount of 5-terminal lacZ mRNA and of -galactosidase activity expressed from these promoters were determined by standard hybridization or enzyme activity assays, respectively. In addition, DNA, RNA and protein measurements were used to obtain information about gene dosage, rRNA synthesis and translation rates. By combininglacZ mRNA hybridization data with gene dosage and rRNA synthesis data, the absolute activity of the different promoters, in transcripts/minute per promoter, was determined. In ppGpp-proficient (relA+spoT+) strains, the respective activities of rrnB P1 and P2 increased 40 and fivefold with increasing growth rate between 0.7 and 3.0 doublings/hour. The activities of Pspc, PL, Pbla, and PRNAIincreased two- to threefold and reached a maximum at growth rates above 2.0 doublings/hour. In contrast, PRNAIIactivity decreased threefold over this range of growth rates. In ppGpp-deficient (relAspoT) bacterial strains, the activities of rrnB P1 and P2 promoters both increased about twofold between 1.6 and 3.0 doublings/hour, whereas the activities of Pspc, PL, Pbla, and PRNAI, and PRNAIIwere about constant. To explain these observations, we suggest that the cellular concentration of free RNA polymerase increases with increasing growth rate; for saturation the P1 and P2 rRNA promoters require a high RNA polymerase concentration that is approached only at the highest growth rates, whereas the other promoters are saturated at lower polymerase concentrations achieved at intermediate growth rates. In addition, the data indicate that the respective rrnB P1 and PRNAIIpromoters were under negative and positive control by ppGpp. This caused a reduced activity ofrrnB P1 and an increased activity of PRNAIIduring slow growth in wild-type (relA+spoT+) relative to ppGpp-deficient (relAspoT) bacterial strains. Copyright 1999 Academic Press Activities of Constitutive Promoters in Escherichia coli ST Liang M Bipatnath YC Xu SL Chen P Dennis M Ehrenberg H Bremer Journal of Molecular Biology (September 1999), pp. 19-37. 2008-06-04T11:53:17-00:00 1999 Journal of Molecular Biology 0022-2836 19 37 Academic Press promoter http://www.citeulike.org/user/msuarezdiez/article/190358 <i>Nucleic Acids Res, Vol. 32, No. 7. (2004), pp. 2147-2157.</i><br /><br />We present a computational method for operon prediction based on a comparative genomics approach. A group of consecutive genes is considered as a candidate operon if both their gene sequences and functions are conserved across several phylogenetically related genomes. In addition, various supporting data for operons are also collected through the application of public domain computer programs, and used in our prediction method. These include the prediction of conserved gene functions, promoter motifs and terminators. An apparent advantage of our approach over other operon prediction methods is that it does not require many experimental data (such as gene expression data and pathway data) as input. This feature makes it applicable to many newly sequenced genomes that do not have extensive experimental information. In order to validate our prediction, we have tested the method on Escherichia coli K12, in which operon structures have been extensively studied, through a comparative analysis against Haemophilus influenzae Rd and Salmonella typhimurium LT2. Our method successfully predicted most of the 237 known operons. After this initial validation, we then applied the method to a newly sequenced and annotated microbial genome, Synechococcus sp. WH8102, through a comparative genome analysis with two other cyanobacterial genomes, Prochlorococcus marinus sp. MED4 and P.marinus sp. MIT9313. Our results are consistent with previously reported results and statistics on operons in the literature. Operon prediction by comparative genomics: an application to the Synechococcus sp. WH8102 genome. X Chen Z Su P Dam B Palenik Y Xu T Jiang Nucleic Acids Res, Vol. 32, No. 7. (2004), pp. 2147-2157. 2005-05-09T20:47:13-00:00 2004 Nucleic Acids Res 1362-4962 32 7 2147 2157 operon prediction