Genetics Exam 4

HIRA

inserts H3 variant H3.3 (actively expressed regions) in nucleosomes near the TSS and in transcribed genes

which pathway is HIRA associated with

associated with transcription dependent pathway

how many times does DNA wrap around a eukaryotic nucleosome?

1\.67

which accessory proteins are required to assist the assembly of nucleosomes and how do they work?

Nucleoplasmin and N1; general chaperones that are acidic and therefore help with wrapping DNA around the the positive histones of the nucleosome

CAF1

recruited to the replication fork by PCNA and it binds with another carrying a dimer to form a new H3/H4 tetramer, which it then adds to newly replicated DNA

what does CAF1 have affinity for?

PCNA, RPA, DNA

Asf1

disassembles parental nucleosomes into H3/H4 dimers; passes new h3-H4 dimers to Caf1

FACT

removes and delivers H2A/H2B dimers from the nucleosome during transcription and DNA replication

histone assembly order

FACT takes off H2A/H2B cap and passes it to asf1, as1 disassembles the nucleosome into H3-H4 dimers, which it passes to caf1. Caf1 joins two H3-H4 complexes to form a tetramer. Caf1 joins DNA due to its affinity for PCNA and mediated the addition of the tetramer into the newly incorporated DNA

Which proteins are associated with replication dependent nucleosome assembly?

Caf1, Asf1, FACT

Which proteins are associated with replication independent nucleosome assembly?

Asf1, FACT, HIRA

The density of nucleosomes in transcribed regions is the same as nontranscribed genes

true

what happens to histone octamers during transcription?

RNA pol displaces them and they reassociate with DNA as soon as the pol has passed

what is used at centromeres instead of H3 in yeast? mammals?

Cse4p; CENP-A

Insulator

defines transcriptionally independent domains by blocking activation and repression (or both), and can block heterochromatin

Insulator in drosophila

scs and scs’ limit the crosstalk between promoters so that high activity regions are near low activity regions; BEAF binds to the scs’

DNA density in bacteria

10mg/ml

DNA density in eukaryotic nucleus

100mg/ml

DNA density in phage T4

500mg/ml

packaging ratio of DNA

length of DNA/length of the unit that contains it

what is the ratio of interphase chromatin? during mitosis?

1000-2000; 5000-10000

length of human DNA

5\.9 ft = 1.8 m

percent DNA in a bacterial nucleotide

\~80%

percent DNA in a eukaryotic nucleus

50%

what is the average density of supercoiling

1 supercoil per 100 bp

what is the size of DNA loops in bacteria

\~100 per genome

Which proteins in E. coli help with DNA packaging?

HU, IHF, H1, protein P

how many DNA loops are in E. coli genome?

40 kb on average

how many loops of DNA in eukaryotes?

\~60 kb (range 20-200kb)

scaffold

proteins that organize chromatin into a condensed state (8% of the protein)

MAR

matrix attachment region; where DNA sequences are bound to the matrix (also associated with scaffold proteins

nuclear matrix

layer of proteins that coats the inside of the nucleus

What two types of proteins have binding sites within MARs?

topisomerases and transctivators

What are the two major components of eukaryotic chromatin?

DNA and histones

What is the fundamental structural unit of chromatin?

nucleosome

euchromatin

transcribed regions that are not densely packed and is dispersed throughout the nucleus

heterochromatin

inactive regions with highly condensed chromatin

constitutive

regions that are not expressed and remain condensed for structural purposes; located near centromeres

facultative

entire chromosomes that are inactive in one cell lineage

example of facultative heterochromatin

mammalian X chromosomes in females

what areas do chromosomes occupy?

specific territories within the nucleus and are not entangled

how is chromatin actually organized?

as a flexible and disordered 5 to 24 nm diameter granular chain without higher order folding patterns

condensin I

forms big main loops at the backbone in condensed chromatin

condensin II

forms smaller nested loops within the larger loops in condensed chromatin

extended state of lampbrush chromosome

found in amphibians during extended mitosis; \~30x less packed than normal; loop is actively transcribed

polytene chromosome

found in drosophila larval salivary gland interphase nuclei; greatly extended packaging ratio; has parallel fibers due to chromosomes doubling up to 9 times

Balbiani rings

transcriptionally active bands are puffed in polytene and chromosome is unwound; site of intense RNA synthesis

metacentric

centromere in the center of sister chromatids

acrocentric

centromeres are off center on sister chromatids

telocentric

centromeres are on the end of the sister chromatids

acentric

does not get attached to mitotic spindle

MTOC (microtubule organizing center)

found at the centrioles at the poles and on each chromosome at the centromere

kinetochore

\~400 nm fibrous structure found at the centromere that is attached to the microtubule during mitosis

Dam1

yeast’s unusual kinetochore

centriole

two clusters of microtubules linked together by other proteins and located at each of the cell poles

centromere

part of the chromosome where microtubules attach and move sister chromatid to cell poles during mitosis

point centromere

budding yeast

regional centromere

insects, mammals, and plants

holocentric centromere

nematode

what do regional centromeres contain?

an alternating pattern of H3 octamers and CenH3 tetramers

Stn1

keeps degradation of C+A strand from the 5’ end from going to far to produce the G+T rich protruding 3’ end

telomerase

extends the 3’OH end of the G+T strand (primer) and contains a covalently attached RNA that serves as a template

telomere

a long series of short tandemly repeated sequences that are 100-1000 repeats

t-loop

3’ single stranded end of telomere TTAGGG invades upstream region and displaces homologous repeats from duplex DNA

what protein is crucial for the T-loop formation?

TRF2 - telomere binding protein that makes a complex with other proteins and stabilizes chromosome ends

Cdc13

binds to telomere repeats and recruits two other proteins, Stn1 and telomerase to trigger the formation of heterochromatin

SIR3/SIR4

binds to the H3 and H4 tails and to the nuclear matrix

RAP1

binds to the DNA and recruits SIR3 and SIR4

How many nucleotides per nucleosome?

200 bp

which part of the histone is most conserved

H3 and H4

How many nucleotides would be left after (high)limit digestion?

146 bp

How many nucleotides would be left after low digestion?

200 bp

What are three common modifications of the histone tails?

Acetylation, methylation, and phosphorylation

HAT

transfer acetyl groups (associated with activation)

HDAC

remove acetyl groups (associate with repression)

what is H3 K9 methylation associated with?

chromatin condensation/repression

what is acetylation associated with?

transcriptional activation

how can drosophila acquire white patches within red eyes?

when the white gene becomes inactive, white patches form and when it becomes active again, red patches form

can eukaryotic RNA pol read DNA?

No, it requires many proteins to prebind to the promoter so that RNA pol can recognize that complex

basal transcription factors

proteins that bind to the core promoter near the start point for transcription and are required to recruit RNA pol

core promoter

region of DNA (including start point of transcription) that binds the basal factors (sequences usually around start and \~100 bp upstream)

start point of transcription

DNA of the promoter that corresponds to the first nucleotide transcribed; RNA pol contacts it directly but doesn’t select it

enhancer

contains several closely arranged/clustered sequence elements that bind transcription factors

what does RNA pol I transcribe?

ribosomal RNAs (18S/28S rRNA)

what does RNA pol II transcribe?

heterogeneous nuclear RNA (protein coding) and few small RNAs

what does RNA pol III transcribe?

tRNA, 5S rRNA and other small RNAs

Rank pols from most activity to least

pol I> pol II> pol III

RNA pol I promoter structure

bipartite; a core promoter and an upstream promoter element (UPE)

RNA pol II promoter structure

lnR and a TATA box OR DPE

RNA pol III promoter structure

Type 1 and 2: internal with boxes

Type 3: upstream with PSE and TATA

which transcription factors are involved with RNA pol I?

SL1 and UBF

which transcription factors are involved with RNA pol II?

TBP (TFIID), TFIIB, A, F, E, H

which transcription factors are involved with RNA pol III?

TFIIIB, A, C, TBP

pol I commitment factor

SL1

pol II commitment factor

TFIID

pol III commitment factor

TFIIIB

how is TBP involved in RNA pol I?

it is a component of SL1

how is TBP involved in RNA pol III?

is a subunit of TFIIIB

what are the assembly factors in RNA pol III?

TFIIIA and TFIIIC

Brf

subunit of TFIIIB that is related to the TFIIB used by RNA pol II