Module 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?
RNAP “explores” and “searches” the genome for promoter sequences. It spends most of its time bound to DNA nonspecifically, and moves in undirected random motion. Stochastic model.
Which DNA strand is part of the DNA-RNA hybrid in the RNA polymerase open complex, the template or nontemplate strand?
The template (antisense) strand.
Explain in general terms how alternative sigma factors can coordinate the expression of multiple bacterial genes or operons.
Alternative sigma factors recognize different promoter sequences. Each sigma factor recognizes a unique -10 and -35 promoter sequence combination. In the cases of regulons, a series of genes controlled by the same promoter sequence, a singular alternative sigma factor controls the expression of all the genes in the regulon.
How do enhancers and super-enhancers interact with promoters spaced 700-1000 bp away?
Transvection hubs
Interactions between enhancers, polymerases, the promoter, insulators, and transcription factors form a transvection “hub.”
Brings the enhancer closer to the promoter, allows the transcription factors to interact with both regions and increase transcriptional activity.
Structure and function of the helix-turn-helix DNA binding motif
Three core alpha helices, the third helix binds directly to the DNA’s major groove
Structure and function of the helix-loop-helix DNA binding motif
Two alpha helices separated by a non-helical loop, doesn’t directly contact the DNA, instead facilitates dimerization of two similar TFs
Structure and function of the basic leucine zipper DNA binding motif
Stretch of amino acids that fold into a long alpha helix, Y-shaped structure. Zipper doesn’t directly contact the DNA, instead facilitates dimerization of two similar TFs
Structure and function of zinc finger DNA binding domain motif
Alpha helices make up the fingers, amino acids between the fingers confer specificity of binding. Alpha helix in the fingers directly contacts the DNA
Which of the four DNA binding motifs directly contact the DNA?
Helix-turn-helix and zinc finger
Which of the four DNA binding motifs do NOT directly contact the DNA?
Helix-loop-helix and basic leucine zipper (instead they both facilitate dimerization of two similar TFs)
Electrophoretic mobility shift assay (EMSA)
Used to study protein-DNA interactions in vitro
Protein-DNA complexes have “shifted mobility”, aka they migrate more slowly, in an electrophoretic field than they do in unbound DNA
Tells us whether a protein binds to a specific fragment of DNA
Does NOT tell us the exact nucleotide sequence with which the protein interacts
Type of gel electrophoresis
ChIP-seq
Used to study protein-DNA interactions in vivo
Proteins are fixed to DNA using either formaldehyde or UV light treatments. The DNA is then sheared into small fragments. The fragments are then incubated with specific primary antibodies to the protein of interest. Incubation steps, yada yada..
If a DNA fragment binds to the protein of interest, it will be recovered in the immunoprecipitate pellet. Any unbound DNA will remain in the supernatant after centrifugation
The DNA is then sequenced
Tells us what DNA sequence a protein of interest will bind to
Enhanceosome model for transcription complex assembly
Proposes that interactions among transcription factors promote their cooperative, stepwise assembly on the DNA and give the complex exceptional stability.
Transcription is regulated by the extensive protein-protein and protein-DNA interactions, and by the context (3D space) in which the elements are organized.
Hit-and-run model for transcription complex assembly
Transcriptional activation reflects the probability that all components required for activation will meet at a certain chromatin site (the hit), transcription complex are assembled in a stochastic fashion from freely diffusible proteins
All the binding in the mess that makes up the “hit” is transient (the run)
What technique has provided evidence for dynamic movement of transcription factors in the nucleus?
FRAP experiments have shown that most nuclear proteins are highly mobile and the interaction of proteins with chromatin and nuclear compartments is highly dynamic. They’ve shown that many of these interactions are quite transient, binding in cycles sometimes as fast as a few seconds. These results lend more towards the hit-and-run model, however the principles of combinatorial interactions and complex stability proposed in the enhanceosome model still apply, even if only for a few seconds.
Definition of epigenetics
Outside of/in addition to genetics
The study of heritable changes in gene expression that occur without a change in the primary DNA sequence of an organism.
Definition of epigenome
The broad array of chemical “marks,” including DNA modifications and post-translational modifications to the histone proteins, that decorate an individual’s genome.
What base in DNA is often methylated in humans?
Cytosine is the most commonly methylated base found in human genomes.
Discuss the role of long noncoding RNAs in X-chromosome inactivation.
X chromosome inactivation is initiated by XIST, a 17kb long noncoding RNA that’s transcribed from a gene located in the X inactivation center.
XIST’s expression is regulated by a series of RNA switches.
XIST is expressed at low levels from both chromosomes before inactivation. When is X inactivation is initiated during differentiation, XIST transcript levels are upregulated from the chromosome that will become the inactive X, while Tsix (a repressor antisense transcript) expression is downregulated
XIST then spreads along the entire length of the X chromosome to be inactivated
Explain to a friend of yours the genetics of coat color in their calico cat.
Coat markings of calico cats are the result of random events during development.
Two alleles affecting coat color are present on the X chromosome, one allele for orange, the other for black. If a female cat is heterozygous for the orange and black alleles, she will have a calico/mosaic coat of orange and black patches.
These patches result from the natural process of X chromosome inactivation that occurs randomly in developing embryos.
In cell lineages, the x chromosome with the orange allele is inactivated, resulting in a patch of black fur, and in others the chromosome with the black allele is inactivated, resulting in a patch of orange fur.
Any white patches of fur are due to an autosomal gene for white spotting, which prevents pigment formation in the cell lineages where it is expressed.
General rule for methylation
Methylation inactivates genes.
Genes with lots of CG methylation are inactive, genes with less CG methylation are active.
CpG islands
CpG islands occur near the 5’ end of genes. They are small regions of CG-rich DNA that are protected from methylation (unmethylated).
Often associated with the promoters of housekeeping genes.
Because CpG islands are protected from methylation, they are also protected from spontaneous deamination
Evidence for cytosine methylation being a host defense mechanism
Because methylated cytosines are more susceptible to C→T deamination than regular cytosine, they aid in the mutation and inactivation of transposable elements, and may also contribute to epigenetic silencing of transposable element transcriptional activity and transposition
Definition of genomic imprinting
Non-random expression of only one of the two alleles inherited from each parent, determined by inherited epigenetic imprints from parental germ cells. This is an exception to the general rule of random selection of the active and inactive alleles, and affects only a small subset of genes.
Describe three general mechanisms for regulating genomic imprinting.
- Altered chromatin structure in the gene promoter
(DNA methylation occurs and repressive chromatin structures form within one allele’s promoter region, silencing that allele) - Differential expression of an antisense RNA transcript (Indirect mechanism. The imprinting control region (ICR) contains the promoter on a non-protein coding gene. On the chromosome where this ICR is unmethylated, the gene is expressed and produces an antisense RNA. This antisense RNA then silences the protein-coding imprinted gene on the same chromosome, while at the same time the methylation of the ICR on the other chromosome ensure that the antisense RNA is not expressed, allowing that other allele to be activated)
- Blocking of an enhancer by an insulator (The ability of shared enhancers (enhancers that control more than one gene) to activate imprinted genes is determined by an insulator present on the unmethylated allele between the genes the enhancer controls)
What are the requirements for gene transcription in bacteria?
The requirements for gene transcription in bacteria are minimal. Only a gene promoter and RNAP are required. However, to regulate transcription, other additional factors are required.
Does RNAP require a primer to get started like DNAP?
No. RNAP can start synthesis de novo.
How does RNAP identify promoter sequences within the vast amounts of chromosomal DNA?
RNAP “explores” and “searches” the genome for promoter sequences. It spends most of its time bound to DNA nonspecifically, and moves in undirected random motion. Stochastic model.
Mobile RNAPs can the whole nucleoid while “searching” for promoters. Is this motion directed or random?
Random
What does it mean when we say that RNAP is shaped like a crab claw?
The 𝛃 and 𝛃’ subunits form structures that look like the pincers of a crab. Additionally, binding of the sigma factor to the core enzyme results in the closing of the pincers and a change in shape of the holoenzyme’s internal channel where transcription takes place. The pincer’s closure contributes to the highly processive nature of the enzyme during transcription elongation.
What other protein does CAP associate with to bind the core RNAP to the promoter?
cAMP
How do coactivators and corepressors differ from transcription factors?
Coactivators and corepressors, unlike transcription factors, don’t directly bind to DNA.
Where on histones do post-translational modifications occur?
On the N-terminal tails of histones H2A, H2B, H3, and H4 in the histone core octamer.
Does HAT repress or activate gene activity? Why?
HAT activates gene activity. HAT attaches negatively charged acetyl groups to histone tail lysine residues, thereby removing the lysine’s positive charge. This decreases the histone tail’s affinity for the DNA, leading to loosened chromatin and increased gene expression.
Does HDAC repress or activate gene activity? Why?
HDAC represses gene activity. HDAC removes negatively charged acetyl groups from histone tail lysine residues, thereby making the histone more positively charged and increasing the histone’s affinity for the DNA, leading to more condensed chromatin and decreased gene expression.
