Lecture 19 & 20

Question 1

What is control over gene expression used for in eukaryotes vs prokaryotes

Answer 1

Prokaryotes – Primarily to allow cells to optimize growth and division in response to changing environments
Eukaryotes – Primarily to regulate a genetic program that underlies embryonic development and tissue
differentiation
– The activating signals for differentiation are transient, but gene expression patterns must be maintained, possibly for decades

Question 2

Gene expression control mechanisms

Answer 2

– The abundance of mRNA is determined by the rate of its synthesis (transcription of the gene),
and the rate of mRNA degradation (mRNA stability)
– The efficiency of translation
– The processing and stability of the protein

Question 3

RNA Pol I promoters (What does it produce, consensus sequences, variation, what level of control)

Answer 3

Large pre-rRNA which is processed into 5.8S, 18S and 28S ribosomal RNAs

Two consensus sequences: UCE and core (CPE)

Very little variation at the promoter

not much control

Question 4

describe the initiation of Pol I Promoter

Answer 4

Initiation occurs when UBF (upstream binding factor) associates with the consensus promoter and SL1

RNA Pol I is recruited, begins to transcribe the pre-rRNA

SL1/UBF complex remains at promoter to recruit another RNA Pol I

Question 5

describe the reinitiation of Pol I

Answer 5

Key to RNA pol I productivity

80%+ of RNA in a cell is rRNA from pol I
In the model system Xenopus laevis (African clawed frog) tandem arrays of rRNA are only 180bp apart
This allows a terminating
RNA pol I to be quickly captured
by the next promoter by the still-bound SL1/UBF complex

Question 6

How is heterochromatin involved in rapid reinitiation

Answer 6

exists as long intergenic spacers between rRNA genes, transcriptionally inactive

Highly repetitive and non-coding
tightly packed

Question 7

Describe RNA Pol III Promoters (diversity, what does it code, what’s unique about it)

Answer 7

More complex and diverse than RNA pol I promoters

RNA pol III specializes in small and highly abundant non-coding RNAs –
tRNAs, 5S rRNA, 7SL RNA, U6 snRNP

uniquely, promoter seq are downstream from the start site

Question 8

how do Promoter vary

Answer 8

vary by which conserved elements they contain

Question 9

Which RNA pol is constitutively expressed with efficient polymerase recycling

Answer 9

RNA pol I

Question 10

How is RNA pol III recruited

Answer 10

The TFIIIC complex recruits the TFIIIB complex which help to recruit RNA pol III to transcribe the short RNA

Question 11

Describe the reinitiation/ recycling of RNA pol III

Answer 11

Recycling of RNA pol III can occur rapidly because TFIIIB remains associated with the upstream DNA

When RNA pol III is pausing at the terminator sequence TFIIIB recaptures RNA pol III

Question 12

Which RNA polymerase has the greatest diversity

Answer 12

RNA polymerase II

Question 13

Which RNA polymerase has the greatest need for control and why

Answer 13

RNA pol II

Some genes need to be up & downregulated across a 10,000-fold range in response to
stimuli
* Some genes are expressed only for a fraction of a percent of the organism’s lifetime &
then never again
* Because of these needs, RNA pol II has the most diverse and complex control mechanisms

Question 14

Which RNA Pol has homologies to bacterial RNA pol

Answer 14

RNA pol II

Question 15

How does RNA pol II resemble bacterial pol

Answer 15

RNA pol II core enzyme has 12 subunits with 5 homologous to bacterial subunits

Question 16

What is the largest subunit in RNA pol II

Answer 16

The largest subunit (RPB1) has many repeats of a 7-aa domain (heptad repeats) at the C terminus which act to control the enzymatic activity

Tyr1–Ser2–Pro3–Thr4-Ser5–Pro6–Ser7

Question 17

What does it mean when a promoter is “strong”

Answer 17

How well any given gene recruits RNA pol II is a product of how “strong” the core promoter is, whether any additional transcription factors assist in the recruiting & the accessibility of the DNA sequence

Whether RNA pol II initiates transcription at any given gene is an issue
of probability

Question 18

What is a consensus sequence

Answer 18

Represents the most-likely base(s) found at each position when looking at promoters

Question 19

what direction is negative and positive

Answer 19

If we consider the first base to be included in a transcript as “+1”, negative labels mean upstream, positive mean downstream

Question 20

What happens when a promoter has a non-consensus base at any position?

Answer 20

It will decrease the likelihood of binding the target protein, which decreases the likelihood of transcription

Question 21

T/F Whole proteins usually contact DNA

Answer 21

Only portions, or domains of a protein

Question 22

Non-specific vs specific interactions

Answer 22

– Nonspecific
* Protein binds DNA (such a histones with basic amino acids binding to phosphate groups by charge interactions)
-Specific
* Protein binds a DNA sequence
* Protein binds a DNA structure

Question 23

describe a series of experiments that can be done to identify where and how a transcription factor can bind DNA

Answer 23

– Step 1: Discover the region of the gene that protein X binds
– Step 2: Show specificity for that gene region
– Step 3: Use that region to determine the exact sequence bound
– Step 4: Determine the structure of the DNA:protein complex

Question 24

describe chromatin immunoprecipitation

Answer 24

Cells expressing our protein are treated to
chemically crosslink any DNA-bound proteins

DNA is then extracted and sheared to fragments ~500nt

Antibodies bound to magnetic beads bind to
the target protein and also the target DNA

Magnetic purification gets rid of all the non-antibody bound DNA

Crosslinking is reversed

DNA is cloned & sequenced

Question 25

Describe DNAse I footprinting. Polyacrylamide vs agarose

Answer 25

DNA fragments that we know bind our protein are amplified using PCR and labelled on only one end of one strand

Purified protein of interest is allowed to bind the target DNA

DNAse I (a randomly cutting endonuclease)
is added, but only long enough that it can cut each PCR product approximately once

The fragments are run on a gel & the absence of bands = the DNA was protected from digestion by the bound protein

agarose can’t tell the difference between 500 and 50 bp but using polyacrylamide, you can tell the difference

Question 26

Describe Electrophoretic Mobility Shift Assay (EMSA)

Answer 26

*Sequence bound by the target protein is labelled
* When run by itself, size is small, band travels to
bottom of gel
* When mixed with protein the band “shifts” up
because the protein:DNA complex is larger
* Mutant probes can be made with alterations to the bound region & made to compete for binding the
limited protein

-if sequence is better, it will bind the protein more readily

Question 27

What does X-ray crystallography do

Answer 27

Proteins, fragments of proteins & protein:DNAcomplexes can be purified and crystallized

Question 28

Advantage of finding the 3d structure using x-ray crystallography

Answer 28

The 3D structure of proteins can reveal which parts are in contact with the DNA & where (backbone, major groove, minor groove)