Study Questions Set 8 Flashcards

1
Q

Describe the role of histone acetylation/deacetylation in regulation of transcription.

A
  • histones are tightly bound to DNA due to the positive charges on the lysine groups, which bind to the negatively charged backbone
  • when histones are acetylated, the positive charges on the lysine groups are neutralized
    o eliminates the tight bond with DNA
    o chromatin gets less condensed and DNA promoters become accessible
  • when histones are deacetylated, DNA will more tightly bind to the histone
    o associated with repression of gene activity
  • histone acetyltransferases are present in co-activators, while histone deacetyltransferases are present in co-repressors
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2
Q

Describe the role of chromatin remodeling complexes in regulation of transcription.

A
  • histone acetylation is essential but not sufficient for activation; nucleosomes are still intact, they have to be repositioned to expose the promoter elements
  • histone octamers may slide along DNA; spacing between histone octamers may be altered producing nucleosome-free gap
  • requires ATP and remodeling complex (e.g., SWI/SNF complex of yeast)
  • (1) SwI/SnF proteins alter nucleosome core structure; (2) Swi proteins move nucleosomes on DNA so that enhancers are in nucleosome-free regions
  • SwI/SnF are not considered transcription factors because they do not actually bind to specific DNA sequences, but are recruited by activators and repressors
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3
Q

Describe an influence of activators and repressors on assembly of initiation complexes.

A
  • Certain TFs need co-activator proteins that do not contact DNA but connect TF with general transcription factors to assemble the initiation complex
  • Repressors can:
    o bind to sites that overlap activator binding sites
    o bind at site adjacent to activator site and interfere with activator
    o bind upstream, interact through mediator with GTFs to inhibit interaction
    o recruit co-repressors that alter nucleosomes in such a way to inhibit transcription
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4
Q

Explain the role of enchancesomes and architectural proteins in regulation of transcription initiation?

A
  • Enhancesome: allows for specific gene expression by interaction at the enhancer; complexes of tissue-specific transcription factors, non-specific transcription factors and co-activators with general transcription factors allows for regulation of various genes across many cell types
  • Architectural Proteins: bind to specific binding sites in order to change the shape of a DNA in a control region so other proteins can interact with each other and/or GTFs and stimulate transcription; it is difficult for DNA to bend/loop if distances between core promoter and enhancer are short, therefore sometimes more than architectural TF is necessary
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5
Q

Explain the role of mediators in regulation of transcription initiation.

A

mediators: proteins that are not in direct contact with DNA, but links transcription factors with general transcription factors; necessary for the assembly of pre-initiation complex
o without them, transcription factors would have no effect on transcription regulation

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6
Q

Explain the role of insulators in regulation of transcription initiation

A
  • insulators: set up boundaries between DNA domains
    o prevents activation/repression of genes by close but unrelated activators and repressors
    o also prevents gene silencing (spreads chromatin modifications – control boundaries between hetero and euchromatin – different on different tissues)
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7
Q

What is the role of DNA methylation in regulation of transcription initiation?

A
  • methylation causes genes to become inactive; methyl groups are attached to C in some CG doublets
    o i.e. CpG islands in promoters of housekeeping genes (genes constantly transcribed)
    o always unmethylated means they are always active
  • methylation prohibits transcription
    o transcription factors can’t bind to promoter if they’re methylated
    o some binding proteins recognize methylated DNA and compete with transcription factors
    o methylation also affects chromatin structure
  • cancer has been linked to global hypomethylation and gene specific hypermethylation
  • during replication of transcriptionally active DNA, associated proteins shield the promoters of active genes from methylation
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8
Q

Describe how steroid hormones regulate transcription.

A

regulate concentration and activity of TFs; important during development
o transcription of transcription factors is regulated many times with extracellular signal hormones (small, lipid soluble molecules, membrane permeable)
o e.g., the steroid hormone Dex is a dependent transport of the steroid hormone receptor that transcribes antibodies to β-Gal

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9
Q

What is the role of transcription factors in development?

A
  • regulation of transcription of transcription factors is highly/strictly regulated
    o there is a critical point at which transcription of genes that produce transcription factors begins
    o dependent on where/when needed; both accessible binding sites and respective binding proteins are necessary for transcription to occur
    o e.g., in Drosophila development, maternal mRNA is already unevenly distributed in embryo
    o when fertilized and cell division occurs, daughter cells inherit different maternal mRNAs
    o certain cells will have different sets of active proteins (some transcription factors – cascade of regulation interactions starts at the very beginning of cell division)
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10
Q

Describe the role of TBP during transcription (think about promoters for all three eukaryotic RNAPs and TATA-less RNAPII promoters – how do RNAPs bind to them; also, think about coordination of activities of all three polymerases).

A
  • TBP is a component of the positioning factor, regardless of the RNAP transcribing; allows each type of RNAP to bind to its promoter
    o coordinates activities of all three polymerases by binding to other polymerase-specific factors
    o is the #1 commitment factor:
     within SL1 complex (RNAP I)
     within TFIID (RNAP II)
     also in TATA-less promoters
     within TFIIIB (RNAP III)
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11
Q

What is the role of Sp1 protein? What is the role of SL1 protein? What do they have in common?

A
  • Both increase the rate of transcription and bind directly to DNA
  • Sp1: transcription factor that binds directly to DNA via a C2H2 zinc finger, enhancing transcription
  • SL1: core-binding factor assembled by UBF-TBP + 3 TBP-associated factors specifically for RNAPI
    o responsible for ensuring RNAP is properly positioned at start point
    o required for high-frequency initiation
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12
Q

What is unusual about type 1 and 2 promoters for RNAP III polymerase?

A
  • They are internal (in reality, changes in regions upstream of start point alter efficiency of transcription)
  • Only when transcription factors bind properly can RNAP III be recruited
  • The details of interaction differ:
    o Type 1: found in genes for 5S rRNA; TFIIIA binds to box A and TFIIIC binds to boxC, enabling TFIIIB to bind at start point, recruiting RNAPIII
    o Type 2: TFIIIC binds to both box A and box B downstream of start point; enables TFIIIB to bind at start point, recruiting RNAPIII
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13
Q

Explain the mechanism of attenuation of the Trp operon. What is the importance of this mechanism for a bacterium?

A
  • attenuation is the regulated, premature termination of transcription
    o does not involve Rho, which is required for transcription termination in 50% of E. coli terminators
    o it is at specific site: the attenuator site (DNA sequence where RNAP either terminates/continues transcription)
    o differs from normal termination, which occurs at the t-site
  • a feedback mechanism that is more directly controlled by molecule of interest (enables the bacteria to conserve resources more strictly)
  • mRNA of trp operon contains 4 regions: region 1 which synthesizes “leader peptide” which is Trp rich, region 2, region 3, and region 4 which is followed by a polyU region; the region 3 and 4 together are called the attenuator
  • chain of events:
    o attenuation (yes or no) depends on formation of the particular stem loop structure in leader sequence
    o formation of the particular stem loop structure depends on rate of ribosomal translation of leader sequence mRNA (connection between transcription and translation – they are going at the same time)
    o rate of ribosomal translation of leader sequence mRNA depends on the supply of tRNA for Trp
    o supply of tRNA for Trp depends on amount of Trp present in cell
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14
Q

Could you imagine a mechanism similar to the mechanism of attenuation of the Trp operon in Eukaryotes? Explain your reasoning.

A
  • no, because the attenuation mechanism involves a formation of a stem loop structure that is creates by DNA in transition between transcription and translation
    o only in prokaryotes can transcription and translation work at the same time on the same mRNA
    o in eukaryotes, transcription and translation work separately (nucleus vs. cytoplasm)
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15
Q

Describe two distinctly different ways in which the trp operon is controlled by the overall availability of tryptophan.

A
  • control in translation:
    o supply of tRNA depends on amount of trp present for ribosomal translation
    o if ribosome stalls at region 1 (requires trp), then stem loop structure forms between 2&3, which allows RNAP to transcribe the subsequent structural genes for trp (because obviously the cell needs more!)
    o if there is plenty of trp, plenty to charge tRNA, ribosome doesn’t stall and instead stem loop forms between 3&4; poly U region follows directly after region 4; triggers RNAP to stop transcription; no structural genes for trp is made, because cell doesn’t need it
  • control in transcription:
    o tryptophan will bind to repressor as a co-repressor
    o enables repressor to make conformation that shuts off transcription
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16
Q

Describe the mechanism responsible for shutdown of the trp operon when a plentiful supply of free tryptophan is available.

A

o RNAP transcribes; nascent mRNA is formed quickly; ribosome will initiate translation at start codon near 5’ end
o regions 1&2 of mRNA may form loop transiently, but no polyU region follows  RNAP pauses, but continues transcribing
o Trp-enriched leader peptide is readily synthesized (stop codon after region 1) – lots of Trp available for ribosome to incorporate rapidly
o Loop involving regions 1 & 2 melts
o Regions 3 & 4 of mRNA form loop –> polyU follows region 4 –> RNAP will terminate transcription before reaching trp structural gene

17
Q

Describe the mechanism responsible for shutdown of the trp operon when there is little tryptophan available

A

o RNAP transcribes; nascent mRNA is formed quickly; ribosome will initiate translation at start codon near 5’ end
o ribosome will stall at Trp codons in region 1 – has to wait for an available Trp-charged tRNA (due to low Trp)
o region 1 of mRNA cannot base-pair with region 2 (is stuck in ribosome) –> region 2 forms loop with region 3 as soon as 3 is synthesized
o no polyU after region 3 –> RNAP continues transcribing –> RNAP elongates past attenuator all the way to “t site” –> structural genes of trp operon are transcribed

18
Q

Describe the mechanism by which the leader-attenuator region fine tunes the extent of transcription of the structural genes in the trp operon when tryptophan is available (but not to the point to activate the repressor).

A
  • when trp is available, ribosome will not stall at region one (which requires tryp)
  • while loop forms between 1&2, it melts quickly and a loop between 3&4 formed instead
  • poly(u) follows region 4, transcription stops at structural genes for tryp
  • this is more temporary shut off/on mechanism; more closely regulates the expression of trp structural genes
19
Q

Describe rho-dependant transcription termination

A
  • rho is a hexameric RNA helices with RNA-binding domain (N-terminal) and ATPase activities (C-terminal)
  • when rho binds to rut site (rho utilization site), it scans along mRNA until reaches RNAP
  • note: rho has time to catch up with RNAP because RNAP has to wait for hairpin to be formed by mRNA for termination site to work properly; rho unwinds RNA and DNA hybrid to release: mRNA, Rho, RNAP
20
Q

Describe rho-independent transcription termination

A
  • Rho-independent termination site has two characteristics:
    o GC rich self-complementary region with several intervening nucleotides that forms stem loop structure
    o Followed by a series of U residues (polyU)
  • after being transcribed, forms loop structure
  • polyU site interacts with RNAP and causes it to pause (it’s a sequence that tells it to slow down)
    o while the polyU region is weakly bonded with complementary A-region on the DNA template, bonds melt as RNAP pauses
    o RNA chain is released
    o DNA strands re-anneal together
    o RNA dissociates from DNA
21
Q

Which proteins are involved in mRNA cleavage and polyadenylation? Describe their order of association with pre-mRNA and their role (don’t forget the role of CTD tail).

A
  • CTD Tail: recruit enzymes necessary for polymerization
  • CPSF: involved in cleavage and polyadenylation specificity factor
  • CStF: cleavage stimulatory factor; binds to G-U region
  • CFI&CFII: cleavage factor I & II bending; mRNA is cleaved
  • PAP-Poly(A) Polymerase binds
  • Slow phase for the first 12 nucleotides
  • Rapid phase requires nuclear polyA binding protein II (PABII)
22
Q

How is 5’ cap added to the nascent RNA?

A
  • added by enzyme that associates with CTD tail of RNAP II soon after RNAPII starts making transcript
    o carried out by mRNA guanyltransferase
    o acts as a barrier to 5’ exonucleases
23
Q

What is the relationship between hnRNA and mRNA?

A
  • hnRNA = heterogeneous nuclear RNA
  • pre-mRNA = primary transcript
  • snRNAs = small nuclear RNA
  • hnRNA = pre-mRNA + snRNAs; transcript that is going to be processed to give mRNAs
24
Q

What are the general steps in processing of a pre-mRNA into an mRNA?

A

1) transcription
2) modification of ends: 5’ capping, addition of 3’ end
3) splicing, exon-intron junctions are broken
4) exons are joined

25
Q

What is the role of snRNAs in the spliceosome?

A
  • snRNAs and their associated proteins form the snRNP which perform spiceosomal splicing
  • the catalytically active components of snRNPs (ribozymes)
  • associated with phosphorylated CTD tail, which:
    o assists snRNAs and Sm proteins in splice site recognition and binding to exons
    o spliceosomal protein-protein interactions
    o conformational changes during spliceosome assembly
    o i.e., U1, U2, U4, U5 and U6; rich in uridine
26
Q

What is the role of Sm proteins in the spliceosome? What other proteins (apart from Sm) are found to be associated with splicing?

A
  • bind “smRNA motif” in snRNAs
  • bind U1, which attaches to 5’ splice site of intron
    o complementarity between intron splice site and snRNA of U1
  • other factors involved in splicing: helicases, proteins that could recognize single-stranded regions
27
Q

What is the difference between splicing of group I and group II introns? Between splicing of group II introns and spliceosomal splicing?

A
  • group I introns: large, self-splicing ribozymes; catalyze their own excision from mRNA, tRNA and rRNA precursors; utilizes GTP
  • group II introns: intron excision occurs in the absence of GTP
    o involves formation of a lariat
    o lariat strongly resembles lariat made during splicing of nuclear pre-mRNAs
  • spliceosomal splicing necessitates the snRNPs subunits and associated protein factors come together; group II introns splice without this complex
28
Q

Describe the current model of spliceosomal splicing.

A
  1. U1 attaches to 5’ splice site of intron (due to complementarity b/w sequences)
  2. U2 binds to branch point A; requires ATP
  3. U4/U6 (base paired with each other) & U5 bind in a tri-complex; binds to 3’ intron splice site; U4 inhibits U6
  4. U4 and U6 change conformation due to the binding, forming B1 complex
  5. U6 dissociates from U4; displaces U1; U1 & U4 are released (ATP dependent)
  6. Once U1 and U4 are released, the spliceosome is activated; it contains U2, U5 and U6 snRNPs (B2 complex); U2-U6 represent active site of spliceosome
  7. U5 is responsible for positioning and holding; U2-U6 complex has catalytic activity, mediates splicing at both sites; formation of C1 and C2 complexes; requires ATP; two trans-esterification reactions
  8. Lariat structure (the pretzel shape) degrades, making splicing irreversible
  9. snRNPs released as individual particles and released
29
Q

List the roles of 5’ methyl cap. List the roles of CTD tail (yes, again).

A

5’ methyl cap:
• recognized by transport machinery
• protection from endonucleases (degradation)
• important in splicing (1st U1 protein binds to 5’ cap)
• ribosome recognizes and binds to 5’ methyl cap for translation initiation

CTD tail:
•	5’ capping
•	polyadenylation: brings enzymes involved
•	splicing
•	initiation of transcription
•	elongation of transcription
30
Q

Describe transcriptional units in Eukaryotes. Use diagrams.

A
  • simple transcription units: when there is only one mRNA product possible; constitutive splicing
  • complex: when there is alternative 3’ exons or alternative internal exons; alternative splicing regulated by specific slicing factors
31
Q

List and explain two means of control of gene expression that could happen during pre-mRNA processing.

A
  • inhibited polyadenylation
  • alternative splicing
    • alternative spliceosome
    • tissue specific RNA splicing control
    • tissue specific alternative poly (A)
    • regulated splicing
  • trans-splicing
  • regulation of mRNA processing
32
Q

Describe one case of control of gene expression by means of splicing.

A
  • alternative splicing where one product may be a functional repressor/inducer, while the other product may be the opposite; these molecules play a role in regulation of gene expression