ch18_lecture.ppt-CSUMBiLearn.ppt

Copyright ?The McGraw-Hill Companies, Inc. Permission required for reproduction or display DNA footprinting was described originally by David Galas and Albert Schmitz in 1978 They identified a DNA site in the lac operon that is bound by the lac repressor This DNA site is, of course, the operator The technical basis for DNA footprinting is this: A segment of DNA that is bound by a protein will be protected from digestion by the enzyme DNase I Figure 18.17 shows a DNA footprinting experiment involving RNA polymerase holoenzyme 18-64 18-65 Figure 18.17 Copyright ?The McGraw-Hill Companies, Inc. Permission required for reproduction or display Did not contain RNA pol holoenzyme Tube A Tube B Labeled end RNA polymerase holoenzyme 150-bp fragment A site where DNase I randomly cuts the fragment A single cut can occur anywhere in the DNA fragment. A single cut can only occur where the protein is not bound. Load onto a gel. Expose the gel to X-ray film. Only the pieces of DNA with a labeled end are detected. Tube A Tube B 150 bases 105 bases 25 bases 1 base +75 +50 +30 +1 –30 –50 –75 Promoter numbering Fragment size Region where RNA polymerase binds Transcription start site 18-66 Figure 18.17 Copyright ?The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the absence of RNA pol holoenzyme, a continuous range of fragment sizes occurs No bands in this range RNA pol holoenzyme is bound to this DNA region, and thus protects it from DNase I Thus RNA pol holoenzyme binds to an 80-nucleotide region (from -50 to +30) Copyright ? The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 18.5 DNA SEQUENCING AND SITE-DIRECTED MUTAGENESIS Analyzing and altering DNA sequences is a powerful approach to understanding genetics A technique called DNA sequencing enables researchers to determine the base sequence of DNA It is one of the most important tools for exploring genetics at the molecular level Another technique known as site-dir