week_8_lecture_2

Made with weaver 2002 molecular biology ed 2 dms

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Unformatted text preview: on) (around • because of large size of DNase, because footprint generated with this enzyme is not high resolution as it is difficult for DNase to gain access to “nooks and crannies”--> gaps in detection of protein-DNA interaction protein-DNA • by DMS footprinting, more by precise analysis of CII contacts with promoter made with Weaver 2002 Molecular Biology Ed. 2 DMS FOOTPRINTING OF PRE & CII or RNAP • DMS footprinting: endlabeled DNA bound by labeled protein protected from protected methylation • methylate with DMS methylate such that about one methylation occurs per DNA fragment DNA • however, if protein however, causes melting of DNA duplex it creates sites more sensitive to more methylation methylation • after methylation, after protein removed,DNA cleaved at methylated sites and run on gel sites Weaver 2002 Molecular Biology Ed. 2 DMS FOOTPRINTING OF PRE & CII or RNAP • detect CII binding to PRE detect RE promoter, protection of bases in the –35 region confirmed confirmed • CII + RNAP footprints CII show RNAP also binds in –35 region but uses different bases • RNAP also binds near – 10 Weaver 2002 Molecular Biology Ed. 2 cII DNASE I -46 -36 -26 -41 -21 RNAP λ LYSOGENY: SUMMARY SUMMARY - - - CII/RNAP binding to PRE CII/RNAP results in “backwards” transcription through cro cro gene and into cI gene, cI thus producing repressor thus CIII protects CII from CIII degradation (CII unstable degradation backwards transcription backwards thru cro gene enroute to cro the cI gene produces cro antisense transcripts that hybridize to cro mRNA, cro inhibiting its translation inhibiting cro inhibition aids lysogeny since cro cro protein necessary for lytic cycle cycle Figure 18-31 λ REPRESSOR - OPERATOR GEOMETRY REPRESSOR N-terminal N-terminal DNA binding domain domain C-terminal C-terminal dimerization domain domain • • • • • Weaver 2002 Molecular Biology Ed. 2 detailed studies of λ detailed repressor: pioneering work on transcriptional regulators inspired work in all organisms (now > 10,000 papers a year on regulation of gene expression!) expression!) 27 kDa repressor subunit = 2 27 domains joined by connector...
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