Exam 2 Flashcards
How does bacterial RNA polymerase find promoters amid vast amounts of chromosomal DNA? What is the role of sigma factor in this process?
RNA polymerase randomly diffuses along the DNA until it finds a promoter it can bind to. Sigma factors help facilitate this process by helping RNA polymerase bind to the DNA. They can target the RNAP to specific promoters.
What DNA strand is part of the DNA-RNA hybrid in the RNA polymerase open complex, the template or non-template strand?
template strand (antisense strand = complementary to mRNA)
Within the bacterial nucleoid, where are highly transcribed genes typically located?
the periphery (provides greater access for RNAP)
For bacterial operons: give specific examples for the following general principles of gene regulation
A. the repressor and activators are DNA binding proteins that undergo allosteric modification
B. cooperative binding of proteins to DNA
A. CAP and the Lac repressor are transcriptional regulators that directly bind DNA. in order for CAP to act as an activator it needs to be bound to cAMP which changes its shape to allow it to bind to the major groove of the DNA double helix. the Lac repressor will stay bound to DNA, inhibiting the expression of the gene until it is bound to glucose, then it will change shape and fall off of the DNA
B. CAP can bind to the DNA and also has a region that can associate with RNAP. in association with a sigma factor, CAP helps RNAP bind tightly to a promoter. If RNAP starts to fall off, CAP is still bound both to it and the DNA, so RNAP is more likely to rebind to the DNA
What is the main difference between Rho-dependent and Rho-independent termination?
Rho-independent: based off of a sequence in DNA that forms an unstable stemloop in the RNA in the RNAP exit channel, this causes RNAP to pause and the transcript is released
Rho-dependent: ribosomes are translating mRNA at the same time as it is transcribed, blocking Rho from loading until the end of the gene. once it is unblocked, rho essentially chases RNAP and knocks it off the DNA
You discover a bacterial RNA that binds an amino acid. In the presence of the amino acid, further expression of the gene that is producing the RNA transcript is blocked. Provide an explanation of how this might be regulated.
This gene’s expression is controlled by a riboswitch. In the presence of the amino acid, the mRNA changes shape to form an alternative structure that closes the reading frame, preventing RNA polymerase from binding
Explain in general terms how alternative sigma factors can coordinate the expression of multiple bacterial genes or operons
Alternative sigma factors can regulate genes w/ promoters specific for particular sigma factor. They can change the overall expression of genes based off of environmental factors. When in a specific environment one alternative sigma factor is more highly available to bind DNA and facilitate more expression of a specific gene.
How do enhancers and super-enhancers interact with promoters spaced 700-1000 bp away?
Physical looping of the chromatin brings them closer together
You suspect that a sequence upstream of a eukaryotic gene transcriptional start site is acting as an enhancer and not as a promoter. Outline an experiment you could run to test your hypothesis.
Create two gene constructs with a reporter gene like lacZ: one where the unknown sequence is close to the start of transcription and one where it is much further away. If there is only expression of the gene when the unknown sequence is close by, then it is a promoter.
Describe how eukaryotic transcription is initiated by the general transcription machinery.
TFIID is the first to bind to the DNA, (recruits the polymerase) then TFIIB signposts the direction and indicates which strand is the template
TFIIH phosphorylates the CTD of RNAP and has helicase activity
What is the function of TFIIH
kinase: phosphorylates the CTD of RNA pol II
helicase: unwinds the DNA
What is the function of mediator
functions as a molecular bridge between long-range regulatory elements and the pre-initiation complex
What roles do HATs, HDACs, HMTs, the SWI/SNF complex, and pioneer factors play in activation/repression of transcription?
HAT - linked to activation of genes - DNA doesn’t wrap as tightly around histone
- HDAC - suppression of genes
HMTs - assoc w/ repression of genes
SWI/SNF - ATP dependent chromatin remodeling, changes the path of DNA around histones
Pioneer factors - TFs that access their DNA target sites in closed chromatin, allowing the binding of other TFs
After initiation of transcription, how does RNA pol II move through nucleosome arrays?
FACT promotes displacement of nucleosomes which allows RNAP to continue to move through the DNA
You are studying a new class of eukaryotic promoters recognized by a novel RNAP. You discover a general transcription factor that is required for initiation of transcription by these promoters. You suspect that it has helicase activity. Outline an experiment you could perform to test your hypothesis.
hybridize a shorter labeled single sequence of DNA w/ a longer single strand
- if it’s not a helicase the two will separate
- if it’s not a helicase the strands will stay together
helix-turn-helix DNA binding motif
two alpha helixes connected by a short turn
- often found in prokaryotic transcription factors
helix-loop-helix DNA binding motif
two alpha helices separated by long, flexible loop
- often found in eukaryotic
leucine zipper DNA binding motif
sequence of AA that folds into long alpha helix with leu every 7th pos - facilitates dimerization
zinc finger DNA binding motif
Cys2-Cys2 pattern stabilized by zinc ions
Electrophoretic mobility shift assay (EMSA)
analyzes in vitro protein-RNA/DNA interactions
if protein binds to a sequence of DNA, then the movement of the sequence will be slower - it won’t move as far in the plate
chromatin immunoprecipitation sequencing (ChiP-seq)
cross-link whole cells with formaldehyde, isolate genomic DNA to produce sheared soluble chromatin, add protein specific antibody, immunoprecipitate and purify immunocomplexes, reverse cross links, purify, prepare for sequencing
You have purified a TF that has a Leu-rich region. You perform an EMSA using a double-stranded oligonucleotide that you know from other studies contains the site recognized by this transcription factor in vivo. However the TF does not bind to the labeled oligonucleotide in your EMSA. Why?
the TF likely has a Leucine zipper which must form a heterodimer in order to bind DNA
Compare the enhanceosome model and hit-and-run model for transcription complex assembly. What technique has provided evidence for dynamic movement of transcription factors in the nucleus?
enhancesome: interactions among TFS promote cooperative stepwise assembly
hit and run: hit - probability that all compenents will meet at a certain site, binding is transient
Define “epigenetics”
the study of heritable changes in gene expression that occur without a change in primary sequence of an organism
What base in DNA is often methylated in humans
cystine
Discuss the role of long noncoding RNAs in X-chromosome inactivation
XIST - lncRNA that coats inactive X
Tsix - lncRNA that represses expression of XIST on active X - activated by another lncRNA
explain the genetics of coat color in calico cats
coat color is on the X chromosome - x-inactivation causes patches of fur that are different colors
Are the majority of CpG islands hypermethylated on the active or inactive X chromosome. Why?
on the inactive X - hypermethylation is associated with inactive genes - block transcription factors from being able to bind to the DNA
Define genomic imprinting
genes that are expressed from only one of the two parental chromosomes - non-random from differential methylation of ICRs
What are the three general mechanisms for regulating genomic imprinting
- differential expression of lncRNA
- blocking an enhancer by an insulator
- altered chromatine structure in gene promoter
How is genomic imprinting “reset” in the germline and maintained throughout development
- global erasure of DNA methylation and histone modification in primordial germ cells
- imprinted genes reacquire marks in spern ad egg - heritable thru division
- another round of demthylation after fertilization but ICRs retain parent specific mehtylation
What would happen during meiosis if imprinting wasn’t reset
might get an egg with a paternal methylation or sperm with maternal, or maybe both alleles are silenced
What does the bisulfate-PCR method distinguish between?
cytosine and 5-methyl-cytosine
If you used the bisulfate PCR method to compare the ICR on chromosome 15 between an unaffected child and one with symptoms of PWS or AS, which chromosome in each case would you expect to see a different pattern
Assume you have two cell-free systems, one for transposition of a DNA transposon, and the other for transposition of a retrotransposon. What effect would the following inhibitors have on the overall process of transposition in these two systems, and why?
A. inhibitors of translation
B. inhibitors of transcription
C. inhibitors of DNA synthesis
D. inhibitors of reverse transcription
Transposition of transposable elements may disrupt genetic function and result in phenotypic variation. Give an example in plants and in humans
plants -
humans -
DNA methylation may serve to defend the genome. Give an example of epigenetic control of a retrotransposon in mice and the resulting mouse phenotypes. Explain how a diet lacking folic acid can lead to activation of the cryptic promoter within this retrotransposon. Is this an example of intergeneration or transgenerational inheritance
intergenerational epigenetic inheritence
transgenerational epigenetic inheritance
some recent animal studies suggest an apparent resistence to complete erasure of epigenetic marks during early development. enabling transgenerational epigenetic inheritence. Whether there are similar mechanisms in humans remains unclear, except for in the case of genomic imprinting. Why is it a challenge to obtain evidence in support of this?
q
List three main characteristics of variant surface glycoprotein gene expression in trypanosomes
What epigenetic mechanisms ensure that only one VSG gene is expressed at a time?
In the most common mode of trypanosome antigen-switching, the VSG gene in the active expression site is degraded and replaced w/ the donor VSG gene. Why isn’t there progressive loss of VSG genes over time.
The VSG gene is transposed through a copy and paste mechanism. So when the gene in the active expression site is degraded and replaced with the donor gene it’s only a copy of the donor gene. The donor gene in it’s original spot in the genome is not degraded.
