Kinetics of Transcription Initiation at lacP1MULTIPLE ROLES OF CYCLIC AMP RECEPTOR PROTEIN*Received for publication, June 6, 2003, and in revised form, July 21, 2003Published, JBC Papers in Press, July 23, 2003, DOI 10.1074/jbc.M305995200Mofang Liu‡§, Geeta Gupte‡, Siddhartha Roy‡¶, Rajiv P. Bandwar储, Smita S. Patel储,and Susan Garges‡From the ‡Laboratory of Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health,Bethesda, Maryland 20892-4264, the¶Department of Biophysics, Bose Institute, P-1/12, C.I.T. Scheme VII M,Calcutta 700 054, India, and the储Department of Biochemistry, Robert Wood Johnson Medical School,Piscataway, New Jersey 08854The cyclic AMP receptor protein (CRP) acts as a tran-scription activator at many promoters of Escherichiacoli. We have examined the kinetics of open complexformation at the lacP1 promoter using tryptophan fluo-rescence of RNA polymerase and DNA fragments with2-aminopurine substituted at specific positions. Apartfrom the closed complex formation and promoter clear-ance, we were able to detect three steps. The first stepafter the closed complex formation leads to a rapid in-crease of 2-aminopurine fluorescence. This was followedby another rapid step in which quenching of tryptophanfluorescence of RNA polymerase was observed. Theslowest step detected by 2-aminopurine fluorescence in-crease is assigned to the final open complex formation.We have found that CRP not only enhances RNA polym-erase binding at the promoter, but also enhances theslowest isomerization step by about 2-fold. Further-more, potassium permanganate probing shows that theconformation of the open complex in the presence ofCRP appears qualitatively and quantitatively differentfrom that in the absence of CRP, suggesting that contactwith RNA polymerase is maintained throughout thetranscription initiation.Most transcription activation occurs during the initiationprocess. Transcription initiation can be divided into a numberof steps (1– 4), and theoretically activation can occur by affect-ing any one of them. Recruitment of RNA polymerase (RNAP)1to a promoter is, of course, the first step in transcription initi-ation, which is followed by a series of isomerization steps.Despite recent availability of a great deal of structural infor-mation on RNA polymerase, elucidation of the process of opencomplex formation remains a major goal. It is difficult to obtainstructural information about these intermediates as they aretransiently populated during transcription initiation. Most ofthe information about the intermediates was obtained indi-rectly, and little is known about the intervening transitionstates. A direct method of studying the kinetics of isomeriza-tion steps would significantly help the dissection of the tran-scription initiation process.The cyclic AMP receptor protein (CRP) can activate the tran-scription of a large number of operons of Escherichia coli (5, 6).To activate transcription, CRP binds to a specific DNA sitelocated upstream from a promoter and interacts with RNAP.CRP activates transcription by using two different modes ofRNAP interaction. In most cases, CRP activates transcriptionfrom a point about 40 bp upstream of the start of transcription,but in others it interacts from a point at least 6 turns of DNAhelix upstream from the transcription start site. The interac-tion points between CRP and RNAP are different depending onwhether CRP is activating from the 40-bp upstream site or theother site. CRP was first identified as a transcription activatorof the lac operon, where CRP binds at a site located 61.5 bpupstream of the start of transcription. From this site, CRPinteracts with the␣subunit of RNAP and helps recruit RNAPto the promoter. Whether the sole function of CRP at the lacpromoter is to help RNAP bind to the promoter remains tobe seen.The kinetics of transcription initiation by CRP at the lacpromoter was examined by Malan et al. (7). By using abortiveinitiation studies, they found that CRP increased the KBofRNAP binding to the lac promoter but had no effect on thesubsequent isomerization step. However, some later resultssuggested effects of CRP beyond the recruitment step, andhence we decided to take a more direct approach and measurethe rates of different steps in isomerization by tryptophanfluorescence and fluorescence of the base analog 2-aminopurine(2-AP) incorporated into the DNA of the lac promoter. In thisarticle we report kinetics of several isomerization steps andinfluence of CRP on each of them.EXPERIMENTAL PROCEDURESPreparation of DNA Templates Containing 2-AP—Both normal andsingle 2-AP incorporated DNA templates were amplified by PCR. First,the ⬃1.1-kb lac operon fragment was amplified from E. coli MG1655genomic DNA. By using this DNA fragment as the template with thesynthetic oligodeoxynucleotides as forward primers (83 bases, Fig. 1),778-bp DNA fragments (78 bp upstream sequence and 700 bp down-stream sequence of the lac operon) containing the lac promoter wereamplified. To abolish the activity of the P2 promoter of lac, a mutationat ⫺29C3 T was introduced at the promoter (8).Separation of PCR DNA Fragment from PCR Mixture—After 4-folddilution by 1⫻ TE buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA), thePCR mixture was loaded on a Resource Q column (Amersham Bio-sciences), which was pre-equilibrated with TE buffer. After loading, thecolumn was washed with 20 ml of TE buffer. Then two gradients (thefirst from 0 to 0.5 M NaCl in 10 ml of TE buffer and the second from 0.5to 0.65 M NaCl in 30 ml of TE buffer) were used to elute the PCR DNAfragment from the Resource Q column at 1 ml/min.Proteins—RNAP was purified from E. coli K-12 cells (MG1655) as* This work was supported by National Institutes of Health GrantGM51966 (to S. S. P.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed: Laboratory of Mo-lecular Biology, NCI, 37 Convent Dr., Rm. 5138, Bethesda, MD 20892-4264. Tel.: 301-451-8820; Fax: 301-496-2212; E-mail: [email protected] abbreviations used are: RNAP, RNA polymerase; 2-AP, 2-ami-nopurine; CRP, cyclic AMP receptor protein; dsDNA, double-strandedDNA.THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 41, Issue of October 10, pp. 39755–39761, 2003Printed in
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