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bmsc320 > Eukaryotic gene transcription > Flashcards

Eukaryotic gene transcription Flashcards

1
Q

What are 6 major differences from prokaryotic transcription?

A
  1. 3 types of RNA pol instead of 1
  2. mRNA undergoes processing
  3. compartmentation of transcription and translation = slower gene expression
  4. more complex regulation
  5. presence of introns
  6. DNA is packaged into chromatin
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2
Q

what is the relationship between the DNA sequence and RNA transcript?

A

RNA will look like non transcript strand aka complementary to the template strand except it will have U instead of T

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3
Q

how do Eukaryotic RNA polymerases differ from DNA polymerases?

A
  1. do not need a primer to initiate nucleic acid synthesis
  2. use nucleotide triphosphates as substrates (NTPs vs dNTPs, UTP instead of dTTP)
  3. Generally slower in catalytic rate
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4
Q

NTPs vs dNTPs

A
  • ribonucleotide is converted to deoxyribonucleotide by nucleotide reductase
  • ribonucleotide has 2 OH groups, deoxyribonucleotide has 1 OH
  • nucleoside is composed of a base and sugar without phosphates
  • nucleotides have at least one phosphate
  • nucleotide triphosphate has 3 phosphates
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5
Q

DNA replication vs RNA transcription

A

Replication: synthesis of both strands, helicase pulls template strands apart, no rewinding after replication fork passes, requires primer

Transcription: synthesis of one strand, RNA pol moves to find initiation site and unwinds DNA, re-winding occurs as the bubble passes, no primer is needed

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6
Q

conservation between prokaryotic and eukaryotic RNA polymerases

A

both have highly conserved cleft domain, diversity occurs on exterior regions

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7
Q

describe the 3 RNA pol in eukaryotes

A

RNA pol I transcribes rRNA
RNA pol III transcribes tRNA, 5S rRNA, and other small RNAs
RNA pol II transcribes mRNA - CTD terminal tail is important

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8
Q

how do things inhibit transcription?

A
  • bind DNA at transcription initiation complex site which prevents elongation of RNA chain by RNA pol II
  • inhibitors can serve as protection mechanisms such as actinomycin D is produced by streptomyces strains
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9
Q

How big are RNA polymerases?

A
  • consist of multiple (11-15) subunits
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10
Q

What is a requirement for eukaryotic RNA polymerases?

A

they can’t identify promoters on their own, so they need other proteins to bind to the promoters first to form a docking site

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11
Q

5 important facts about RNA pol I

A
  1. transcribes only one type of gene: rRNA gene (but NOT 5S rRNA)
  2. accounts for 50% of transcriptional activity
  3. rRNA gene is transcribed and processed into three RNA products: 18S, 5.8S, and 28S RNA
  4. Organization of the rRNA gene is conserved across different species
  5. there are 200 copies of the rRNA gene in most genomes arranged in tandem repeats
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12
Q

Structure of eukaryotic ribosomes

A
  • made up of large and small subunit (which are made separately)
  • 40S and 60S make 80S (not additive)
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13
Q

characteristics of human genomic rRNA variants

A
  • variant rRNA alleles give rise to physically and functionally heterogenous ribosomes that contribute to pathology and human disease
  • mutations can alter how quickly ribosomes make proteins or can make ribosomes work better
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14
Q

5 basic parts of rRNA gene transcription and processing

A
  1. transcriptional units occur in tandem (number of repeats varies)
  2. Only one strand is template/transcribed, the other is non-template
  3. Transcript is produced that still contains spacers
  4. Spacers are removed (akin to intron splicing)
  5. Mature rRNA molecules are generated (18S, 5.8S, and 28S)
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15
Q

where does transcription of rRNA gene take place?

A
  • in the nucleolus (inside nucleus)
  • each small nucleolar ribonuclear protein (snoRP) complex includes enzymes that modify nucleotides, ribosomal proteins, snoRPs, and exo/endo-ribonucleases
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16
Q

what are the 4 steps of RNA modification/folding?

A
  1. 90S pre-ribosome is modified by methylation or pseudouridine formation
  2. initial cleavage of pre-rRNA into pre-40S and pre-60S
  3. additional cleavages and export from nucleus to cytoplasm
  4. final maturation occurs to produce 40S and 60S rRNAs
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17
Q

What is the role of promoters (4 points)?

A
  • define where transcription starts
  • determine direction of transcription
  • determine rate of transcription
  • determine regulatory properties of transcription of the particular gene
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18
Q

What does frequency of initiation depend on?

A

strength of the promoter
- if weak, sends a few pols spaced out
- if strong, sends many pols close together
highly transcribed = strong promoter

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19
Q

3 steps of assembly of the Pol 1 Preinitiation complex (PIC)

A
  1. upstream binding factor (UBF) binds upstream control elements (UCE) and core element within rDNA promoter
  2. Stably bound UBF recruits SL-1 initiation factor which is composed of many TBP associated factors (TAFs)
  3. Stable UBF-SL-1 complex recruits initiation competent RNA pol I in an interaction mediated by RRN3
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20
Q

How does positioning of elements affect rate of PIC assembly?

A

if way upstream, will be slower
if closer to start, will be faster

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21
Q

what is the purpose of rRNA gene promoter-looping

A

compensates for the large distances between mammalian upstream control elements and core elements
- if UCE and core elements are too far apart to form a dimer, looping the DNA in between them brings them close enough to form dimer

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22
Q

What is SL1

A
  • a multi-subunit protein
  • consists of TBP (TATA binding protein) and TAFs (TBP associated factors)
  • TBP + TAF complexes are used by all 3 eukaryotic RNA pols, differing only in type of TAFs and number included
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23
Q

how are RNA pol I molecules continuously engaged in transcription

A
  • linking termination and re-initiation on tandem gene repeats
  • 2 mechanisms depending on species
  • xenopus (frog): RNA pol I terminates only 180 bps away from the next promoter, next promoter “grabs” RNA pol I and re-engages it in transcription of next tandem gene. Tandem array facilitates active re-engagement of RNA pol I
  • mammals: long spacer region can be up to 30,000 bp in length, so looping spacer regions brings termination site and next promoter into close proximity and the next promoter can grab RNA pol I
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24
Q

What is the function of RNA pol III?

A

transcribe genes coding for all tRNAs, 5S rRNA, many snRNAs through polygenic sequence

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25
Q

What are the 3 types of RNA pol III PICs?

A

Type I: (5S rRNA) promoter occurs within gene
Type II: (tRNA) promoter occurs within gene
Type III: (U6 RNA component for splicing) promoter occurs upstream of gene

26
Q

what are the 3 promoters recognized by RNA pol III?

A
  1. tRNA genes (transfer RNA; protein translation)
  2. 5S rRNA genes (ribosome biogenesis)
  3. U6 snRNA genes (intron splicing)
27
Q

structure of tRNA gene promoter

A
  • internal promoter downstream of the TSS
  • consists of boxes A and B
  • ordered sequential binding to form docking site
  • TFIIIC binds boxes A and B within transcript (not within promoter) then recruits TFIIIB (made up of TBP and 2 TAFs: BDP1 and BRF1) which recruits RNA pol III
  • no TATA box
28
Q

Structure of 5S rRNA gene promoter

A
  • contains internal promoter site downstream of the TSS consisting of boxes A and C
  • TFIIIA binds box C, which recruits TFIIIC at box A
  • leads to recruitment of TFIIIB and pol III
  • no TATA box
29
Q

structure of U6 snRNA gene promoter

A
  • contains canonical promoter site upstream of the TSS consisting of TATA box and proximal sequence elements (PSEs)
  • TBP binds TATA box while snRNA-activating protein complex (SNAPC) binds PSEs which recruits dfifferent form of TFIIIB (With BRF2 instead of BRF1) to transcribe U6 snRNA
30
Q

what is the function of RNA pol II?

A
  • responsible for transcribing all protein-coding genes and many microRNA (miRNA) genes
31
Q

why is pol II most complicated?

A
  • transcribes a different transcriptome in each cell in order to have cellular differentiation
  • mRNA product also undergoes significant processing in nucleus before being transported to cytosol and translated into protein
32
Q

What are the 4 steps of the eukaryotic transcription cycle?

A
  1. start with DNA and RNA pol
  2. PIC is built in DNA to start initiation
  3. RNA pol II binds and begins transcription until it reaches termination site
  4. RNA is released and pol is released to start a new cycle
33
Q

6 steps of PIC assembly on a TATA box containing promoter

A
  1. TBP (found in TFIIB) is recruited to TATA box
  2. TFIIA gets recruited
  3. TFIIB gets recruited (rate limiting event)
  4. RNA pol II is recruited with TFIIF
  5. The unphosphorylated CTD tail on pol II is incorporated into PIC
  6. TFIIE gets recruited (rate limiting event) and TFIIH gets recruited and phosphorylates the CTD tail which releases it from the PIC
34
Q

what does TBP do when bound to TATA box?

A
  • binds within minor groove which bends DNA and causes it to open up, allowing access to the other factors
  • brings proteins bound within the promoter into closer proximity
35
Q

What does recruiting TFIIB do to DNA?

A
  • causes zig zag distortion of the DNA helix
    forms a pocket in TFIIA
36
Q

How is TBP part of the TFIID complex?

A
  • TFIID binds to TATA box through TBP subunit
  • TBP binds to the minor groove of DNA
37
Q

3 major roles of TAFs (TBP associated factor)

A
  1. activation (histone acetyltransferase (HAT) is associated with gene transcription)
  2. TFIID tethering to non-TATA promoters
  3. Promoter security
38
Q

What are the two general types of promoters recognized by RNA pol II?

A

Type 1 (25%): contain TATA boxes at position -25
Type 2 (75%): do not contain TATA box

39
Q

Characteristics of type 1 RNA pol II promoters

A
  • contain TATA box at -25
  • have initiator sequence around TSS
  • tend to be highly expressed genes
40
Q

characteristics of Type 2 RNA pol II promoters

A
  • do not contain TATA box
  • tend to be housekeeping genes
  • subtype A contains initiator sequence around the transcription start site YYANA/TTY, and a downstream sequence
  • subtype B has no initiator sequence but has a GC rich promoter with no distinct transcription start site
41
Q

How does TBP interact with a TATA-less promoter?

A
  • TATA-less promoter with initiator motif (Inr) and downstream promoter element (DPE): TFIID uses other factors to bind to Inr and DPE, TBP is recruited with the complex
  • TATA-less promoter with GC boxes: TFIID uses Sp1 to recognize GC sequence, TBP is recruited with the complex
42
Q

What is a unique feature of the large subunit of RNA pol II that is critical to gene expression and regulation?

A
  • carboxy terminal domain (CTD)
  • consists of repeating amino acid sequence forming heptad repeats:
    (Tyr/Ser/Pro/Thr/Ser/Pro/Ser)n aka (Y/S/P/T/S/P/S)n
  • humans n= 52; drosophilia n = 44; yeast n = 26
  • Tyr/ser/thr can be phosphorylated at various times and place
  • proline provides bends and kinks to polypeptide chain
43
Q

what is important about heptad repeats?

A
  • they are all similar and highly conserved, but not identical
  • any protein that interacts with DNA will be conserved from yeast to humans (even though the CTD doesn’t directly interact with DNA, it interacts with transcription factors that do bind DNA and are highly conserved, so CTD will be conserved as a consequence)
  • different heptad repeats serve distinct functions (N-terminal half supports only capping of mRNA; C-terminal half supports capping, splicing, 3’ end processing of mRNA)
44
Q

what is function of CTD?

A
  • binding scaffold for proteins involved in mRNA processing
  • strategically located next to exit channel for mRNA
  • when phosphorylated it links stage of transcription with RNA processing, elongation, and termination
  • phosphorylation affects recruitment of chromatin re-modifying enzymes
45
Q

What are 2 possible modifications of CTD?

A
  1. Tyr and Ser all have OH groups than can be modified by phosphorylation which changes structure and function of amino acids (on/off)
  2. Isomerases change isomer of amino acids - depending on cis or trans conformation they will have different functions
46
Q

4 important facts about the CTD

A
  1. mutant RNA pol II that lack CTD transcribe genes at a slow but unregulated rate and cannot be activated
  2. CTD is highly de/phosphorylatable (only unphosphorylated form can enter PIC, phosphorylated form transcribes the gene)
  3. unphosphorylated form physically binds TBP, keeps RNA pol II locked at promoter
  4. phosphorylation pattern of CTD can differ from gene to gene and changes as RNA pol II proceeds down the gene
47
Q

what 3 things known about dynamic CTD phosphorylation pattern?

A
  1. Ser5 phosphorylation predominates near beginning of genes
  2. Ser2 phosphorylation predominates near end of genes
  3. Tyr1 phosphorylation increases downstream of initiation site, decreases before poly-A site, impairs recruitment of termination factors, and recruits elongation factors
48
Q

Principle of CTD patterns

A
  • modifications both trigger and block recruitment to generate CTD code
  • explains transcription cycle coordination based on differential phosphorylation of Tyr1/Ser2/Ser5
  • different motifs are recruited in different proteins
49
Q

What are possibilities of CTD code?

A
  • S2 phosphorylated first (and only)
  • S5 phosphorylated alone
  • S2 and S5 phosphorylated
  • since CTD can have up to 52 heptads there are many different combos/codes
50
Q

Structure and function of TFIIE and TFIIH

A
  • TFIIH consists of five subunits, one has helicase activity which helps melt DNA and allow formation of transcription bubble, one has kinase activity and can phosphorylate the CTD of RNA pol II at Ser 5
  • TFIIE stimulates the kinase and helicase activities of TFIIH
51
Q

What is the trigger hypothesis?

A
  1. RNA pol II CTD wraps around the the entire complex - assembled but not active
  2. TFIIH kinase domain phosphorylates the CTD tail
  3. CTD tail releases the entire complex and is actively transcribing

TBP is important to create a bend in DNA that allowsTFIIB to bind PIC which causes another bend that brings in TFIIA

52
Q

How do transcription factors mediate pol II transcription?

A
  • long distance effects of enhancers occur by DNA bending
  • silencers repress transcription
  • insulators restrict transcription to a single loop in a chromosome
53
Q

What is Mediator?

A

multi-subunit complex recruited to PIC that links enhancers with the PIC

54
Q

what two things do most transcription factors have?

A

DNA binding domain/motif (DBD) and transcriptional activation motif (ACT)

55
Q

How does the mediator work?

A
  • links an activator protein (bound to enhancer sequence) to RNA pol II
  • can link multiple transcription factors at once by looping the DNA, effect is the same gene can be transcribed in different ways depending on which TFs bind to the mediator
  • different subunits of Mediator function in specific biologic processes, so if missing subunits, it will not function in certain processes
  • has many subunits (might not all be used all the time) so that in cases of stress where an immediate response is needed, they can start right away instead of waiting for the TFs to get recruited
56
Q

important notes on mediator

A
  • enhances recruitment of RNA pol II and other initiation factors
  • acts as a bridge between DNA-bound TFs and PIC
  • can enhance basal transcription in absence of bound activators
  • individual components can have gene-specific and tissue specific functions
  • several forms exist that can activate or repress transcription depending on which factors are present in mediator
57
Q

what are characteristics of transcription factors?

A
  • can be activators or repressors
  • directly bind to specific DNA sequence or response element
  • specific TFs have at least 2 functional domains (DNA-binding domain DBD; Transcription activating domain ACT) that are independently folded and often function separately which often bind DNA as dimers
58
Q

what are the three classes of transcription activating domains?

A
  1. Acid domains (regions rich in Glu and Asp)
  2. Glutamine-rich domains
  3. Proline-rich romains
59
Q

specific transcription factor: cAMP response element binding protein (CREB)

A
  • found in genes whose transcription is induced by cAMP
  • common conserved sequence in promoters called cAMP response element (CRE): 5’ TGACGTCA 3’
    1. when glucose levels drop, induces release of glucagon which increases cAMP
    2. cAMP activates protein kinase A (PKA)
    3. PKA finds and phosphorylates CREB which is bound to CRE in nucleus
    4. Phosphorylated CREB recruits CBP
    5. CBP increases rate of transcription of genes required to make glucose
60
Q

specific transcription factor: SREBP - regulation of transcription by cholesterol

A
  • cholesterol is necessary for membrane synthesis and is precursor for steroid hormones/bile acids
  • decreased cholesterol causes genes whose protein products increase cholesterol to be turned on
  • these genes all possess a sterol response element (SRE) in their promoters, the protein that binds to this sequence is SREBP

-when cholesterol is low, SREBP is transported to the golgi where it is processed by two proteases into active form which turns on target genes
- when cholesterol is high, SREBP is kept in the ER, not processed, and does not activate genes
- SREBP cleavage activating domain (SCAP) is a sterol sensor that escorts SREBP to the golgi

61
Q

specific transcription factor: steroid hormones

A
  • cortisone reduces inflammation by altering transcription
    1. TNFα induces inflammation, causes binding of NFₖB to IₖB
    2. IₖB is phosphorylated and degrades which frees NFₖB to move to nucleus
    3. NFₖB binds target gene promoters that release cytokines
    4. Cytokines drive inflammation and trigger the release of cortisone
    5. cortisone interacts with glucocorticoid receptor to be able to bind to IₖB gene promoter which increases transcription of IₖB
    6. Increased IₖB binds to NFₖB which inhibits NFₖB from acting

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