BMB 400 Part Three-II = Chpt. 11. Transcription: Promoters and TerminatorsB M B 400, Part ThreeGene Expression and Protein SynthesisChapter 11. TRANSCRIPTION: PROMOTERS, TERMINATORS ANDmRNAThis second chapter on transcription focusses on the cis-acting elements needed foraccurate transcription, with a emphasis on promoters. The chapter begins with a discussion oftechniques used to find the start site for transcription and to identify the segments of DNA boundby protein. It then covers promoters, elongation, termination, and mRNA structure. Thephenomenon of polarity is explored to show the relationships among mRNA structure, transcriptionand translation in E. coli.A. Mapping the 5' ends of mRNAThe nucleotide in DNA that encodes the 5' end of mRNA is almost always the startsite for transcription. Thus methods to map the 5’ end of the mRNA are criticalfirst steps in defining the promoter.Figure 3.2.1. Nuclease protection to map 5’ end of a gene1. "S1 protection assay"This assay measures the distance between an end label (at a specific known site on DNA)and the end of a duplex between RNA and the labeled DNA. A fragment of DNA(complementary to the RNA) that extends beyond the 5' end of the RNA is labeled at arestriction site within the RNA-complementary region. The labeled DNA is hybridized toRNA and then digested with the single-strand specific nuclease S1. The resulting fragmentof protected DNA is run on a denaturing gel to determine its size. Note that this fragmentruns from the labeled site to the nearest interruption between the DNA and the RNA. Thiscould be the beginning of the RNA, or it could be an intron, or it could be an S1 sensitivesite.BMB 400 Part Three-II = Chpt. 11. Transcription: Promoters and TerminatorsFig. 3.2.2. Nuclease protection assay to define the 3’ end of a gene.2. "Primer extension assay"This assay measures the distance between an end label and the point to whichreverse transcriptase can copy the RNA. A short fragment of DNA, complementaryto RNA, shorter than the RNA and labeled at the 5' end, is hybridized to the RNA.It will now serve as a primer for synthesis of the complementary DNA by reversetranscriptase. The size of the resulting primer extension product gives the distancefrom the labeled site to the 5' end of the RNA (or to the nearest block to reversetranscriptase).Fig. 3.2.3. Primer extension assay, another way to map the 5’ ends of genesBMB 400 Part Three-II = Chpt. 11. Transcription: Promoters and Terminators3. How do you label DNA at the ends?a. 5' end label: T4 polynucleotide kinase and [γ 32P] ATP. The reaction ismost efficient if the 5' phosphate is removed (by alkaline phosphatase) priorto the kinase treatment.b. 3' end label: Klenow DNA polymerase plus [α 32P] dNTP. The labeleddNTP is chosen to be complementary to the first position past the primer. Arestriction fragment with a 5' overhang is ideal for this "fill-in" labeling.c. Digestion with a second restriction endonuclease will frequently work toremove the label at the "other" end. One can also use electrophoretic gelsthat separate strands.4. A PCR-based technique to determine the 5’ ends of mRNAs and genesA technique utilizing the high sensitivity of PCR has been developed to determine the 5’ends of mRNAs which can then be mapped onto genomic DNA sequences to find the5’ ends of genes. This technique is called rapid amplification of cDNA ends and isabbreviated RACE. When RACE is used to determine the 5’ end of mRNA, it is called5’ RACE. This method requires that an artificial primer binding site be added to the 5’ends of copies of mRNA, or cDNA, and knowledge of a specific sequence within thecDNA, which will serve as the second, specific primer for amplification during PCR(Fig. 3.2.3b).Fig. 3.2.3.b. Rapid amplification of cDNA ends, or 5’ RACEBMB 400 Part Three-II = Chpt. 11. Transcription: Promoters and TerminatorsThe methods for making cDNA from mRNA are more prone to copy the 3’ ends andmiddle of mRNAs than the 5’ ends. Thus it is common to have access to this part ofthe cDNA, and that provides the sequence information for the second, or internal,primer. In contrast, specialized techniques are often employed to get information aboutthe 5’ ends of mRNAs. In the technique outlined in Fig. 3.2.3.b, the fact that reversetranscriptase tends to add a few C residues to the 3’ end of the cDNA is used to designan artificial template that will anneal to those extra C nucleotides. Then reversetranscriptase copies the second template, thereby adding the artificial primer bindingsite. This artificial primer binding site is needed because the sequence of the 5’ end ofthe mRNA is not known in this experiment; indeed, that is what the experimenter istrying to determine. Once the artificial primer binding site has been added to thecDNA, then the modified cDNA serves as the template for PCR. The PCR product issequenced and compared to an appropriate genomic DNA sequence. The first exon orexons of the genes will match the sequence of the PCR product, starting right after thefirst primer.B. General methods for identifying the site for sequence-specific binding proteins1. Does a protein bind to a particular region?a. Electrophoretic mobility shift assay (EMSA), or gel retardation assayThis assay will test for the ability of a particular sequence to form a complexwith a protein. Many protein-DNA complexes are sufficiently stable thatthey will remain together during electrophoresis through a (nondenaturing)polyacrylamide gel. A selected restriction fragment or synthetic duplexoligonucleotide is labeled (to make a probe) and mixed with a protein (orcrude mixture of proteins). If the DNA fragment binds to the protein, thecomplex will migrate much slower in the gel than does the free probe; itmoves with roughly the mobility of the bound protein. The presence of aslowly moving signal is indicative of a complex between the DNA probe andsome protein(s). By incubating the probe and proteins in the presence ofincreasing amounts of competitor DNA fragments, one can test forspecificity and even glean some information about the identity of the bindingprotein.BMB 400 Part Three-II = Chpt. 11. Transcription: Promoters and TerminatorsFigure 3.2.4. Diagram of results from an electrophoretic mobility shift assayComplex AComplex BFree ProbeExtractSelfE. coliSp1 Oct1CompetitorLane___++ ++ + + + + + + + + +12 3 45 6 78 9 10 11 12 1314In this example, two proteins