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Molecular Genetics > Histones and Nucleosomes > Flashcards

Histones and Nucleosomes Flashcards

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1
Q

How many nucleotides per nucleosome?

A

200 bp

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2
Q

How many nucleotides would be left after limit digestion (maximum, extreme digestion)?

A

146 bp

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3
Q

Which histone is not present in the 10 nm fiber and is required to form the 30 nm fiber?

A

H1

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4
Q

Which histones comprise the eukaryotic nucleosome?

A

H3, H4, H2A, H2B

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5
Q

Which histones are the most ancient and can be found in Archaea?

A

H3 and H4

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6
Q

How does the Archaeal nucleosome differ from eukaryotic nucleosomes?

A

Archaeal nucleosome is a tetramer and the eukaryotic is an octomer of two copies each of H4/H3 and H2A/H2B

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7
Q

Do Archaeal histones have tails? Why or why not?

A

does not have to have a histone code to control condensation of chromatin

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8
Q

explain how histones are related to 10 nm fibers

A
  • composed of dsDNA and nucleosome
  • beads on a string structure
  • fundamental unit of structure for the chromosome
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9
Q

what is the general structure of the nucleosome?

A
  • 200 bp of DNA
  • two copies of each core histone, H2A and H2B form a dimer at the ends while H3 and H4 form a tetramer at the core
  • histone fold (helix - turn - helix - turn - helix) located at the core
  • histone tails at N terminal of both faces of the nucleosome
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10
Q

what is the structure of histone tails?

A
  • N terminal
  • flexible and disordered
  • exit from both faces of the nucleosome between turns of DNA
  • no set structure, no loops, or alpha helices
  • site of the histone code
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11
Q

what is the difference between HAT and HDAC?

A
  • HAT = histone acetyltransferase, associated with activation and adds acetyl groups
  • HDAC = histone deactylase, associated with repression and removes acetyl groups
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12
Q

chromatin structure

A
  • has compact organization in which most DNA sequences are structurally inaccessible and functionally inactive
  • 60% protein (half = histones), 30 % DNA, and 10% RNA
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13
Q

how much DNA is recovered in nucleosomes or multimers when micrococcal nuclease cleaves DNA of chromatin?

A

more than 95%
- the length of DNA per nucleosome varies for individual tissues in a ranfe from 154 - 260 bp with an average of 200 bp

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14
Q

how is nucleosomal DNA divided?

A
  • core DNA and linker DNA
  • depends on the DNA’s susceptibility to micrococcal nuclease
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15
Q

low DNase (nicking)

A
  • 200 bp
  • mono-nucleosomes
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16
Q

high DNase (limit digestion)

A
  • 146 bp
  • core particles
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17
Q

what would the average size of the fragments of chromatin be if you added just enough nuclease to hit once per nucleosome?

A

200 bp

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18
Q

how many times does DNA wrap around the histone octamer?

A

1.67 times
- 165 bp

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19
Q

what are the most conserved proteins known?

A

H3 and H4

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20
Q

which proteins have species specific variations in sequence?

A
  • H2A and H2B
  • H1 but also tissue species specific variability
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21
Q

what is the role of histone H1 in chromatin?

A
  • can form pre-nucleosome with mammalian H3/H4
  • not in archaea
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22
Q

which proteins “cap” the two flat ends of the core nucleosome?

A

H2A and H2B

23
Q

what are the types of modifications that occur with histone tails?

A
  • acetylation
  • methylation (methylases)
  • phosphorylation (kinases)
  • some modifications reduce the positive charge of the histone
24
Q

what does acetylation do to the positive charge in histones?

A

neutralize it

25
Q

does mono and di-methylation change the positive charge in histones?

A

do not not neutralize the positive charge, so no

26
Q

what does phosphorylation do to histones?

A

introduces negative charges on serine

27
Q

what occurs to lysine and arginine in methylation and acetylation in histones?

A
  • the positive charge on lysine is neutralized upon acetylation
  • methylated lysine and arginine retain their positive charges
28
Q

what is the histone code?

A

pattern of modifications present on the tails of histones H3 and H4 that determines the activation state of genes

29
Q

what do H3 histones do? H4?

A
  • directly affect transcription state
  • directly affect nucleosome assembly
30
Q

H3 lysine 4

A

methylation - activation

31
Q

H3 lysine 9

A
  • methylation - repression
  • chromatin condensation
  • acetylation - activation
  • transcription activation
32
Q

H3 serine 10

A

phosphorylation - transcription activation

33
Q

H3 lysine 14

A

acetylation - prevents methylation at lys-9

34
Q

H4 arginine 3

A

methylation - affects nucleosome assembly

35
Q

H4 lysine 5

A

acetylation - affects nucleosome assembly

36
Q

H4 lysine 12

A

acetylation - affects nucleosome assembly

37
Q

what happens during acetylation?

A
  • acetylation during replication occurs on specific sites on histones before they are incorporated into nucleosomes
  • acetylation is associated with gene activation and occurs directly modifying specific sites on histones that are already incorporated into nucleosomes
38
Q

which enzyme removes acetyl groups from histons?

A

HDAC

39
Q

what are the two pathways for nucleosome assembly?

A
  • replication dependent (replication fork) and replication independent (transcription, repair)
40
Q

which two proteins act as accessory proteins?

A
  • nucleoplasmin and N1
  • acidic and function as a general chaperons and may assist in wrapping DNA around nucleosome
41
Q

what is the order in which histones are assembled?

A
  • CAF1 binds to new H3/4 tetramer then H2A/B is added
  • nucleosome is added as soon as new DNA appears
42
Q

what is the role of CAF1 and ASF1?

A
  • used in replication dependent assembly
  • ASF1 and CAF1 act as a chaperone
  • CAF1 recruited to replication fork by PCNA (associated with alpha and epsilon)
  • replication fork displaces histone octamers from DNA
  • ASF1 feeds new H3/4 dimers to CAF1 while CAF loads it as a tetramer onto new DNA
  • FACT disassembles the nucleosome, ASF1 binds to new H3/4 for lagging strand
43
Q

what is the role of FACT?

A
  • ancillary factor
  • acts to displace octamers during transcription by releasing H2A and B dimers, other proteins needed to re-assemble after transcription
  • FACT participates in re-assembly after RNA pol passes
44
Q

what is the role of HIRA?

A
  • inserts H3 variant H3.1 in nucleosomes near he TSS and in transcribed regions
  • occurs during transcription and in cells not undergoing replication
45
Q

When are the histone 3 (H3) variants used?

A
  • histone 3.3 slowly replaces H3 in non-differentiating cells and after transcription
  • also used in repair
  • centromeric DNA replicates early and uses Cse4p in yeast or CENP-A in mammals instead of H3 - does not use replication dependent pathway
46
Q

What is the role of an “Insulator”?

A
  • define transcriptionally independent domains
  • an enhancer activates a promoter in its vicinity but can be blocked from doing so by an insulator located between them
  • can block activation, repression, or both
  • may block heterochromatin
47
Q

BEAF

A
  • heat shock loci 87A and C of drosophila are flanked by insulators
  • a protein that binds to the insulator scs’ and is localized interbands in drosophila polytene chromosomes
48
Q

specialized chromatin structures (scs and scs’)

A
  • do not directly regulate gene activity but function to limit the crosstalk between promoters so that regions of very high activity can be located next to low activity regions of the genome
49
Q

do RNA pol 2 and insulator proteins occupy the same regions?

A

they do not

50
Q

what part of the histone is not conserved during replication?

A

histone ocramers
- H2A and H2B dimers and H3-4 tetramers are conserved

51
Q

what is the structure of CAF1?

A
  • 5 subunits
  • mass of 238 kD
52
Q

when are nucleosomes found at the same frequency when you digest with micrococcal nucleoase?

A
  • transcribed genes
  • nontranscribed genes
  • some heavily transcribed genes appear to be exceptional cases that are devoid of nucleosomes
53
Q

are histone octamers displaced by transcription?

A
  • RNA polymerase displaces histone octamers during transcription in a model system
  • octamers reassociate with DNA as soon as the polymerase has passed
  • nucleosomes are reorganized when transcription passes through a gene
54
Q

what are the relative sizes of RNA polymerase and nucleosomes?

A
  • nucleosome = 300 kb, 6x11 nm
  • RNA pol 2 = 500 kb, 14x13

Molecular Genetics (28 decks)

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