lf206 lecture 5 - eukaryotic genomes, transcriptional regulation

why is transcriptional regulation needed?

allows development of different tissues

transition from childhood to adult

allows reaction to environmental cues

deregulation can result in uncontrolled growth → cancers

what determines how/when genes are transcribed? [3]

1. chromatin structure

2. RNA polymerase binding specificity

3. additional binding and activation factors

facultative heterochromatin have H3K27me3 (__________) which is a histone modifaction.

H3K27me3 can bind to ________ proteins. these proteins remodel ________.

trimethylation of histone 3 at lysine 27

polycomb

chromatin

constitutive heterochromatin are regions that are consistently ________

silenced

this is a eukaryotic nucleus. actively expressed genes (green) are central. heterochromatic regions (red) are close to the nuclear membrane.

what does this suggest?

DNA can be moved to the nuclear membrane for transcriptional inactivation

centric heterochromatin have long highly _________ chromatin structures.

_________ allows for an open structure, permitting kinetochore (define this) attachment

repetitive

H3K27me3

kinetochore binds chromosome to mitotic spindle aligning to metaphase plate

telomeres are a repeated ___ sequence on the ends of DNA

DNA

COMPENSATORY MECHANISM FOR TELOMERE SHORTENING

• telomeres have a single-stranded ______-rich overhang

1. telomerase binds to the G-overhand

2. telomerase is a __________ (RNP) enzyme made of telomerase ___ (TER) and telomerase reverse ________ protein (TERT)

3. ________ extends the __’ end of the parental strand using its own __ subunit as a template.

4. this is a reverse _______ process.

5. free 3’ unpaired end triggers repair __________

6. RNA-templated DNA synthesis by _______ extends the G-overhang 5’-3’, DNA _______ lays down RNA _____ on the extended G overhang

7. DNA-templated DNA synthesis by ___ ________ extends this primer 5’-3’, ___ ligase ligates the new _______ fragment to the old lagging strand 5’ end

1. guanine

2. ribonucleoprotein, RNA, transcriptase

3. telomerase, 3’, RNA

4. transcription

5. mechanisms

6. telomerase, primase, primer

7. DNA polymerase, DNA, Okazaki

3’ end of telomere extension needs to shelter from repair mechanisms.

why and how?

it resembles DNA damage

shelterin complex of TRF1 (telomeric repeat-binding factor 1), TRF2 and RAP1 (repressor/activator protein) stimulates t-loop formation.

this displaces a d-loop and results in base pairing on 3’ end.

if telomeres cannot be replicated what can this lead to?

Werner syndrome - premature aging symptoms

___ binds to the TATA box on RNA polymerase ___, and the strength of the binding regulates ________

TBP (TATA-binding protein)

II

transcription

TATA box is a consensus sequence.

what does this mean?

TATA boxes have different ________ for TBP and so, some are more efficient at stimulating _________ than others.

a DNA sequence pattern that appears repeatedly in different places across genes

not identical but common pattern

affinities

transcription

G-less cassette transcription assay

1. a promoter is cloned upstream of a _-less cassette

   1. add purified transcription factors, RNA Pol __, ATP, CTP and [alpha^32P]-UTP (what is special about this?)

   2. produces a radioactive ___ transcript which can be _________ through polyacrylamide gels and quantified following autoradiography

principle: because no ________ (GTP), RNA is truncated where _ should be

G

II

it is radioactive

RNA

electrophoresed

guanosine triphosphate