Lecture Molecular biology (Fifth Edition): Chapter 13 - Robert F. Weaver

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Chapter 13 - Chromatin structure and its effects on transcription. In this chapter, we will look at the crucial relationship among activators, chromatin structure, and gene activity. This chapter presents the following content: Histones, nucleosomes, chromatin structure and gene activity.

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Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 13 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 13 Chromatin Structure and Its Effects on Transcription Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chromatin Structure • Eukaryotic genes do not exist naturally as naked DNA, or even as DNA molecules bound only to transcription factors • They are complexed with an equal mass of other proteins to form chromatin • Chromatin is variable and the variations play an enormous role in chromatin structure and in the control of gene expression 13-2
13.1 Histones • Eukaryotic cells contain 5 kinds of histones – H1 – H2A – H2B – H3 – H4 • Histone proteins are not homogenous due to: – Gene reiteration – Posttranslational modification 13-3
Properties of Histones • Abundant proteins whose mass in nuclei nearly equals that of DNA • Pronounced positive charge at neutral pH • Most are well-conserved from one species to another • Not single copy genes, repeated many times – Some copies are identical – Others are quite different – H4 has only had 2 variants ever reported 13-4
13.2 Nucleosomes • Chromosomes are long, thin molecules that will tangle if not carefully folded • Folding occurs in several ways • First order of folding is the nucleosome, which have a core of histones, around which DNA winds – X-ray diffraction has shown strong repeats of structure at 100Å intervals – This spacing approximates the nucleosome spaced at 110Å intervals 13-5
Histones in the Nucleosome • Chemical cross-linking in solution: – H3 to H4 – H2A to H2B • H3 and H4 exist as a tetramer (H3-H4) 2 • Chromatin is composed of roughly equal masses of DNA and histones – Corresponds to 1 histone octamer per 200 bp of DNA – Octamer composed of: • 2 each H2A, H2B, H3, H4 • 1 each H1 13-6
H1 and Chromatin • Treatment of chromatin with trypsin or high salt buffer removes histone H1 • This treatment leaves chromatin looking like “beads-on-a-string” • The beads named nucleosomes – Core histones form a ball with DNA wrapped around the outside – DNA on outside minimizes amount of DNA bending – H1 also lies on the outside of the nucleosome 13-7
Nucleosome Structure • Central (H3-H4)2 core attached to H2A- H2B dimers • Grooves on surface define a left-hand helical ramp – a path for DNA winding – DNA winds almost twice around the histone core condensing DNA length by 6- to 7-X – Core histones contain a histone fold: • 3 -helices linked by 2 loops • Extended tail of abut 28% of core histone mass • Tails are unstructured 13-8
Crystal Structure of a Nucleosomal Core Particle 13-9
The 30-nm Fiber • Second order of chromatin folding produces a fiber 30 nm in diameter – The string of nucleosomes condenses to form the 30-nm fiber in a solution of increasing ionic strength – This condensation results in another six- to seven-fold condensation of the nucleosome itself • Four nucleosomes condensing into the 30- nm fiber form a zig-zag structure 13-10
Models for the 30-nm Fiber • The solenoid and the two-start double helix model each have experimental support • A technique called single-molecule force spectroscopy was employed to answer the question, ‘which model is correct?’ • Results suggested that most of the chromatin in a cell (presumably inactive) adopts a solenoid shape while a minor fraction (potentially active) forms a 30-nm fiber according to the two-start double helix 13-11
Higher Order Chromatin Folding • 30-nm fibers account for most of chromatin in a typical interphase nucleus • Further folding is required in structures such as the mitotic chromosomes • Model favored for such higher order folding is a series of radial loops Source: Adapted from Marsden, M.P.F. and U.K. Laemmli, Metaphase chromosome structure: Evidence of a radial loop model. Cell 17:856, 1979. 13-12
Relaxing Supercoiling in Chromatin Loops • When histones are removed, 30-nm fibers and nucleosomes disappear • Leaves supercoiled DNA duplex • Helical turns are superhelices, not ordinary double helix • DNA is nicked to relax 13-13
13.3 Chromatin Structure and Gene Activity • Histones, especially H1, have a repressive effect on gene activity in vitro • Histones play a predominant role as regulators of genetic activity and are not just purely structural • The regulatory functions of histones have recently been elucidated 13-14
Effects of Histones on Transcription of Class II Genes • Core histones assemble nucleosome cores on naked DNA • Transcription of reconstituted chromatin with an average of 1 nucleosome / 200 bp DNA exhibits 75% repression relative to naked DNA • Remaining 25% is due to promoter sites not covered by nucleosome cores 13-15
Histone H1 and Transcription • Histone H1 causes further repression of template activity, in addition to that of core histones • H1 repression can be counteracted by transcription factors • Sp1 and GAL4 act as both: – Antirepressors preventing histone repressions – Transcription activators • GAGA factor: – Binds to GA-rich sequences in the Krüppel promoter – An antirepressor – preventing repression by histones 13-16
A Model of Transcriptional Activation 13-17
Nucleosome Positioning • Model of activation and antirepression asserts that transcription factors can cause antirepression by: – Removing nucleosomes that obscure the promoter – Preventing initial nucleosome binding to the promoter • Both actions are forms of nucleosome positioning – activators force nucleosomes to take up positions around, not within, promoters 13-18
Nucleosome-Free Zones • Nucleosome positioning would result in nucleosome-free zones in the control regions of active genes • Assessment in SV40 DNA, a circular minichromosome, was performed to determine the existence of nucleosome-free zones - with the use of restriction sites it was found that the late control region is nucleosome free 13-19
Detecting DNase-Hypersensitive Regions • Active genes tend to have DNase-hypersensitive control regions • Part of this hypersensitivity is due to absence of nucleosomes 13-20