Slide 1Role of H1 (cont.)What is the effect of histones on transcription in vitro?In Vivo Studies3 ways to a clear (or nucleosome-deficient) promoterSlide 6What about elongation by RNA polymerase?Experimental StrategySlide 9Slide 10Slide 11Slide 12Equation for SupercoilingSlide 14Slide 15Slide 16+ H1- H1The role of histone H1H1 binds to the nucleosome where the DNA enters and exits the core.5 mM NaCl1 mM NaCl H1 is needed to form the zig-zag structure.Role of H1 (cont.)•Evidence indicates that H1 also functions in forming the 30 nm fiber, and that it interacts with other H1 molecules H1 histoneDNANucleosomecoreH1-H1 interaction proven by cross-linking experiments.What is the effect of histones on transcription in vitro? •Assemble core histones on a plasmid (1/200 bp), nucleosomes inhibit transcription by blocking promoter binding sites. •Addition of H1 further represses transcription (by binding to the linker DNA), but this can be overcome by activators such as Sp1.•There are regulatory proteins, such as the glucocorticoid-receptor complex, that can remove histones from certain promoters.In Vivo Studies •Promoters of active genes are often deficient in nucleosomesSV40 virus minichromosomes with a nucleosome-free zone at its twin promoters. From Fig. 13.17Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I.3 ways to a clear (or nucleosome-deficient) promoter 1. Transcription factor(s) (for the respective RNA polymerase) bind the promoter before histones– there is competition to bind newly synthesized DNA2. If H1, which binds linker DNA, is blocking the promoter, it can be displaced by some transcription factors (e.g., SP1).3. If a nucleosome does form on the key part of the promoter, it has to be removed by special complexes to get transcriptional activation - a.k.a. “chromatin remodeling” .2 Models for Transcriptional Activation H1 (yellow) covers promoter, remove it and bind activators (factors).Nucleosome covers promoter, still repressed after H1 removed. Remove nucleosome with special “remodeling” factors.Similar toFig. 13.16What about elongation by RNA polymerase?•How does RNA polymerase transcribe through regions with nucleosomes?•2 possibilities: 1. It could partially open nucleosomes and slide around the DNA, which is on the outside.Or 2. It could completely displace nucleosomes.Experimental Strategy1. Construct DNA with only 1 nucleosome on it, downstream of a promoter.2. Transcribe DNA in vitro.3. Determine if the nucleosome moves to a new position*.•* DNA specifically bound in a nucleosome can be recovered after Micrococcal Nuclease digestion – which degrades all DNA not protected by the nucleosome.Conclusion: some of the nucleosome (blue oval) is repositioned on pB18 after transcription in vitro with the viral RNA polymerase. Fig. 13.43Fig. 13.44(a) Restriction map of pB18, and (b) expected DNA fragments with different enzyme combinations.DNA protected by the nucleosome core is recovered after micrococcal nuclease digestion, radiolabeled, and then hybridized to restriction enzyme digests of pB18.Eukaryotic chromosomal DNA alsohas supercoiled regions.Fig. 13.10Positive Supercoiling or OverwindingTwist the strands further around one another in the same direction as the coiling of thehelix. This will cause the helix to become more tightly wound.3’Right handedWrithing structuresWrithing – double helical strand passing over itselfNegative Supercoiling or UnderwindingTwist the DNA in the opposite direction to the helix's coil. This loosens the winding ofthe single strands. If you keep doing this you will reduce the base pairing.Writhing structuresEquation for SupercoilingLk = Tw + WrLk = Linking number (# times that strands cross)Tw = Twist (# of times that strands cross, excluding writhe, or # of helical turns)Wr = Writhe (# of times that double helix crosses itself)Topoisomerase I relaxes DNA one link at a timeReduces Lk one unit at a time.+ Topo IType I Topoisomerases changeLn by 1, breaks 1 strand.Type II Topoisomerases – change L by 2, cuts both
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