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METHOD 1. Set up in vitro transcription reaction with DNA template, 32P-UTP, plus or minus transcription factor SP1 2. Separate transcripts by gel electrophoresis QUESTIONS: 1. Name the DNA for which transcription requires SP1. 2. What is the evidence as seen in the autoradiogram? 3. What type of transcription factor is SP1? QUESTIONS: Based on the results shown in the autoradiogram, we see a band in lane 1 (from transfection with plasmid 1) with but not in lane 2 (from transfection with plasmid 2). 1. What does the band in the autoradiogram represent? 2. What do these results tell us about the nature of the extra piece of DNA included in plasmid 1 and absent in plasmid 2? (What is the “name” for this extra piece of DNA?) Lecture 9 Transcription Termination, mRNA processing, & posttranscriptional control Reading: Chapter 12 Molecular Biology syllabus web site Transcription termination • Several mechanisms exist to regulate the termination of transcription in bacteria and eukaryotic cells • In bacteria, the two principle mechanisms involve RNA polymerase and one of these also requires the termination factor Rho • In eukaryotes, the mechanisms for terminating transcription differ for each of the three types of RNA polymerase Rho-independent termination occurs at characteristic sequences in E. coli DNA Premature termination by attenuation helps regulate expression of some bacterial operons Mechanism of attenuation of trp-operon transcription Rho-dependent termination sites are present in some -phage and E. coli genes • The Rho factor is a hexameric protein around which a 70- to 80-base segment of the growing RNA transcript wraps • Rho then moves along the RNA in the 3 direction until it eventually unwinds the RNA-DNA hybrid at the active site of RNA polymerase • Whether transcription is terminated or not depends on whether Rho “catches up” to RNA polymerase • Rho-dependent sites have no clear consensus sequence and Rho-dependent termination operates at relatively few operons Figure 11-4 Three eukaryotic RNA polymerases employ different termination mechanisms • RNA polymerase I is terminated by a mechanism that requires a polymerase-specific termination factor, which binds downstream of the transcription unit • RNA polymerase II is terminated in a region 0.5-2 kb beyond the poly(A) addition site, and termination is coupled to the process that cleaves and polyadenylates the 3 end of a transcript • RNA polymerase III is terminated after polymerizing a series of U residues Transcription of HIV genome is regulated by an antitermination mechanism Processing of eukaryotic mRNA The 5-cap is added to nascent RNAs after initiation by RNA polymerase II Pre-mRNAs are cleaved at specific 3 sites and rapidly polyadenylated During the final step in formation of mature, functional mRNA, introns are removed and exons are spliced together Splicing occurs at short, conserved sequences Consensus sequences around 5 and 3 splice sites in vertebrate pre-mRNA Analysis of RNA products formed in an in vitro splicing reaction Splicing proceeds via two sequential transesterfication reactions Small nuclear RNAs (snRNAs) assist in the splicing reaction Self-splicing introns-evolutionary models of trans-acting snRNAs? Other post-transcriptional regulatory mechanisms • Alternative splicing (e.g. ion channels affecting auditory cells; affecting wiring/neuronal connections in the brain) • mRNA 3’ ends that target mRNA to cytoplasmic location (resulting in protein gradients across cell) • mRNA stability affected by 3’ untranslated sequences • Regulation of antisense transcripts and siRNA • RNA editing (common in mt in protozoa/plants and chloroplasts; rarer in higher eukaryotes) • Post-transcriptional modification (e.g. tRNA) • Translational activation of mRNAs (cytoplasmic polyadenylation of stored mRNAs with short polyA tailsinduced upon fertilization of xenopus oocytes or synaptic activity in neuronal dendrites)