Lecture 19 & 20 Flashcards
What is control over gene expression used for in eukaryotes vs prokaryotes
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
Gene expression control mechanisms
– 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
RNA Pol I promoters (What does it produce, consensus sequences, variation, what level of control)
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
describe the initiation of Pol I Promoter
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
describe the reinitiation of Pol I
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
How is heterochromatin involved in rapid reinitiation
exists as long intergenic spacers between rRNA genes, transcriptionally inactive
Highly repetitive and non-coding
tightly packed
Describe RNA Pol III Promoters (diversity, what does it code, what’s unique about it)
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
how do Promoter vary
vary by which conserved elements they contain
Which RNA pol is constitutively expressed with efficient polymerase recycling
RNA pol I
How is RNA pol III recruited
The TFIIIC complex recruits the TFIIIB complex which help to recruit RNA pol III to transcribe the short RNA
Describe the reinitiation/ recycling of RNA pol III
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
Which RNA polymerase has the greatest diversity
RNA polymerase II
Which RNA polymerase has the greatest need for control and why
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
Which RNA Pol has homologies to bacterial RNA pol
RNA pol II
How does RNA pol II resemble bacterial pol
RNA pol II core enzyme has 12 subunits with 5 homologous to bacterial subunits
What is the largest subunit in RNA pol II
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
What does it mean when a promoter is “strong”
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
What is a consensus sequence
Represents the most-likely base(s) found at each position when looking at promoters
what direction is negative and positive
If we consider the first base to be included in a transcript as “+1”, negative labels mean upstream, positive mean downstream
What happens when a promoter has a non-consensus base at any position?
It will decrease the likelihood of binding the target protein, which decreases the likelihood of transcription
T/F Whole proteins usually contact DNA
Only portions, or domains of a protein
Non-specific vs specific interactions
– 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
describe a series of experiments that can be done to identify where and how a transcription factor can bind DNA
– 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
describe chromatin immunoprecipitation
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
Describe DNAse I footprinting. Polyacrylamide vs agarose
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
Describe Electrophoretic Mobility Shift Assay (EMSA)
*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
What does X-ray crystallography do
Proteins, fragments of proteins & protein:DNAcomplexes can be purified and crystallized
Advantage of finding the 3d structure using x-ray crystallography
The 3D structure of proteins can reveal which parts are in contact with the DNA & where (backbone, major groove, minor groove)
