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MHG 1 > Transcription I, II, III > Flashcards

Transcription I, II, III Flashcards

1
Q

DNA strands

A
  1. Nontemplate = coding = positive 5’ → 3’

*RNA transcript matches coding strand

  1. Template = non-coding = negative 3’ → 5’
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2
Q

Prokaryotic Polymerase

&

Eukaryotic Polymerase II

subunits

A
  1. Core enzymes: ßß’ω
  2. 2α subunits
  3. σ subunit is variable. Brings polymerase to promoter and dissociates when txn starts
    * MW dependent; σ70
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3
Q

Other Eukaryotic RNA polymerases

A
  1. Pol I: synthesizes the transcript that is the precursor for rRNAs 18S, 5.8S, 28S
    * rRNA processing involves methylation
  2. Pol III: synthesizes tRNAs and the 5S rRNA
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4
Q

Steps of prokaryotic transcription (4)

A
  1. Binding: RNA Pol holoenzyme-thru σ subunit binds to -35 promoter region → forms closed complex
  2. Initiation: Pol migrates to -10 promoter region → “melting” ds → open complex → 90o angle →txn starts w/ a purine→ σ subunit leaves
  3. Elongation: RNA-DNA hybrid forms as dNTP is added to 3’OH in active site releasing PPi
    * Txn bubble: positive supercoils ahead and negative supercoils behind relieved by topoisomerase
  4. Termination: complementary sequences
  • ρ-dependent: hairpin structure forms and pol pauses. ρ protein causes dissociation
  • ρ-independent: hairpin structure followed by stretch of Us. Hairpin destroys hybrid b/c A-U binding is unstable
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5
Q

Negative regulation

A

Bound repressor inhibits txn

  • Associates to operator region in promoter
    1. Binding of molcular signal causes repressor dissociation from DNA → allows txn
    2. Binding of molecular signal causes binding of repressor on DNA → inhibit txn
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6
Q

Positive regulation

A

Bound activator facilitates txn

  • Associates to operator
    1. Binding of molecular signal causes activator dissociation from DNA → inhibit txn
    2. Binding of molecular signal causes activator binding to DNA → allow txn
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7
Q

LAC OPERON

in

high glucose, low lactose levels

A

TURNED OFF

  1. Gene I encodes for the lac repressor under the control of the PI promoter
  2. Lac repressor binds to operator sites O1 and O2
  3. Causes dimerization → DNA loops
  4. Prevents txn of genes Z Y A
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8
Q

LAC OPERON

high lactose, low glucose

A

TURNED ON

  1. Low glucose → increase in cAMP

cAMP binds to CRP in CRP site upstream of promoter

  1. lactose → Allalactose binds to repressor to cause dissociation from promoter

Pol is recruited to promoter to stabilize interaction *consensus sequence is not ideal, requires help

  1. TXN of Z: ß-galactosidase / Y: ß-galactoside permease / Z: ß-galactosid transacetylase
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9
Q

ARA OPERON

high glucose, low arabinose

A

TURNED OFF

  1. AraC synthesized by auto regulation
  2. AraC binds to AraI and AraO2
  3. Causes AraC dimerization: forms loop
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10
Q

ARA OPERON

L-Arabinose present

A

TURNED ON

  1. Upon arabinose binding to AraC–> AraC dimer dissociates (conformational change) → activator
  2. Low glucose → increase cAMP → binds to CRP → brings RNA pol to bind to promoter Pc
  3. TXN: BAD genes → metabolize arabinose
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11
Q

TRP OPERON

high levels of Tryptophan-tRNA

A

ATTENUATED

  1. Ribosome quickly translates mRNA sequence before RNA polymerase transcribes sequence 3 (covers sequence 2)
  2. 3:4 complementary pair → termination signal due to hairpin & string of Us
  3. RNA polymerase dissociates and stops txn
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12
Q

TRP OPERON

Free tryptophan levels

A

HIGH TRP: TURNED OFF

  1. Trp binds to repressor
  2. Trp-Repressor complex binds to operator

LOW TRP: TURNED ON

  1. Repressor dissociated from operator
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13
Q

TRP OPERON

Low levels of Tryptophan-tRNA

A

TURNED ON

  1. Ribosome pauses at TRP codons in sequence 1
  2. RNA pol continues transcription → 2:3 complementary → txn continues
  3. Genes for Trp synthesis: E, D, C, B, A
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14
Q

Promoter elements for RNA polymerase

(general)

A
  • 10 region & -35 region
    1. Greater consenus → better RNA pol binding → higher transcriptional activity
  • Method in which houskeeping or constituitive genes maintain level of gene expression
    2. Induction of activity due to stimuli
  • under different stress conditions, different σ factors will be expressed to activate different operons
  • heat shock protein
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15
Q

Eukaryotic promoter elements

A
  1. Locations: far upstream, within introns, within exons, 3’ end of coding region
  2. “cis” regions are conserved regions = binding sites for transcription factors (trans factors) are often in **distal enhancer or repressor regions **
  • time & stimuli dependent
  • enhancers are position & orientation independent
  1. Proximal promoter: TATA box (-30) - TBP binds (TFIID) CAT box (-70 to -90) - contains GC box - Sp1 binds

*Genes that lack TATA and CAT are often rich in GC → multiple binding sites for Sp1; multiple start sites (common in housekeeping genes → alwasy on)

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16
Q

Pre-initiation complex

@ TATA box

A
  1. TFIID recognizes TATA box via TBP
  2. TFIIB binds to TBP & recognized by TFIIF
  3. TFIIA stabilizes binding
  4. TFIIF & RNA polymerase II TFIIF dissociates after 1st base added
  5. TFIIE recruits TFIIH & ATPase & helicase activities
  6. TFIIH- phosphorylate Pol II, proofreadng fxn by recruiting NER proteins, helicase activity
  7. TFIIJ
17
Q

Repressor mechanisms (5)

A

Transcription factors of negative regulation

  1. Competition: interacts at same or overlapping site
  2. Activator sequestering: direct interaction with activator
  3. Activator masking: indirect - interact with DNA
  4. Silencing: chromatin condensation via methylation
  5. Locking: interact with initiation complex
18
Q

Activator mechanisms (6)

A
  1. Protein synthsis: when needed
  2. Ligand binding: GR, ER, AR, PR
  3. Phosphorylation: AP2 txn factors (GF signaling pthway)
  4. Addition of 2nd subunit
  5. Unmaking: inhibitor leaves - retinoblastoma binds E2F
  6. Stimulation of nuclear entry - degradation of inhibitory protein - IF kappa B and NF kappa B

*Can directly disassemble nucleosomes and facilitate assembly of preinitiation complex and txn factors

19
Q

Transcription factors structural properties

A
  1. Helix-turn-helix motif common in bacteria
  2. Zinc finger common in eukaryotic
    * Steroid hormone - Nuclear receptors
  3. Leucine zipper: bind as dimer
  • amphipathic α helix with Leu residues every 7 AA forming a straight line on hydrophobic side; helices wrap: coiled coil
  • Lys and Arg rich in DNA binding region
  1. Basic helix-loop-helix in eukaryotic
  • short amphipathic α helices; basic AA
  • txn factor MAX and NIC
  1. Structural domains important for protein-protein interactions with RNA pol: glutamine rich domains, proline rich domains, acidic activation domains
    * GAL4 stabilizes TFIID and TFIIB in yeast
20
Q

Inhibitors of transcription (4)

A
  1. actinomycin D: intercalates in DNA btwn CG bases and blocks RNA pol; inhibits elongation (*antibiotic)
  2. Rimfampicin: binds to ß subunit of prokaryotic pol
  3. α-amanitin (mushrooms): blocks eukaryotic pol II
  4. acridine: intercalates DNA
21
Q

Chromatin regulation of eukaryotic txn (4)

A
  1. ACTIVE-beads on a string: increased sensitivity to nucleases & hypersensitive sites in promoter regions
  2. Locus control regions (LCR) contains hypersensitive sites, binds multiple regulatory proteins, and is involved in generating active chromatin structure
    * Mutations abolish all txn from entire domain
  3. Nucleosome remodelings: HATS and HDACS
    * SWI/SNF hydrolyze ATP and move nucleosomes to allow txn
  4. Methylation of promoter CpG regions
22
Q

RNA processing: Capping

(6 steps)

A
  • . Occurs during txn
  • . Addition of: 7-methyl-guanosine residue
  • . Linked by: 5’-5’ triphosphate bond
    1. TFIIH phosphorylates C terminal end of RNA pol → binding site for Cap-synthesizing complex
    2. phosphoydydrolase removes γ phopshate
    3. GTP and guanyly transferase add GMP & release PPi
    4. adoMet and guanine-7-methyltransferase
    5. adoMet and 2’-O-methyltransferase (only in vertebrates)
  • in basic conditions, OH can cause 2’OH on sugar to act as nucleophiles and attack phosphodiester backbone
  • +water → 2’ or 3’ nucleotide
  1. after Cap synthesizing complex leaves, CBC binds to pol II to keep 5’ anchored to polymerase
23
Q

Capping & poly A tail purposes

A
  1. Allows mRNA exit from nucleus
  2. Aids in efficient transcription
  3. Maintains half-life
  4. Protection from exonucleases
24
Q

RNA processing: Splicing

Self-splicing method 1

A

Occurs in protazoans, yeast, fungi

Intron region bounded by 5’-UA and 3’-GU

  1. 3’OH of guanosine (GTP, GDP, GMP) acts as a nucleophile, attacking 5’ phosphate of A
  2. Exposed 3’OH of U acts as nucleophile, attacking 5’ phosphate of U → recreate phosphodiester bond

*Intron: pGpA ——- pG-OH

25
Q

RNA processing: Splicing

Self-splicing method 2

A

*intron: G —-pCpApA——

  1. 2’OH of Adenosine acts as a nucleophile, attacking 5’ phosphate on G
  2. Lariat structure: 2’-5’-phosphodiester bond tethered to 3’ end
  3. 3’OH of exposed U acts as a nucleophile, attacking 5’ phosphate on U → recreates phosphodiester bond
26
Q

RNA processing: Splicing

Eukaryotic splicing (method 3)

4 steps

A

snRNP (small nuclear ribonucleoproteins) - made of snRNA and proteins

Intron: 5’ GU ——–A—AG 3’

  1. U1 → U2 *ATP → U4/U6 + U5 *ATP → inactive spliceosome
  2. U1 and U4 dissociate * ATP → active spliceosome
  3. donor jxn broken & 2’OH of A binds 5’ phosphate on G → lariat structure
    * Binding of U2 - bp causes A to bulge out
  4. acceptor jxn broken & exons ligated together

intron released with snRNPs

*spliceosome binding site created at phosphorylated regions on CTD polymerase; occurs while RNA is transcribed

27
Q

ß thalosemia

A
  1. Normally, α and ß globin proteins are synthesized 1:1
  2. Mutation in branch point causes U2 to sometimes recognize ß globin
  3. ß globin synthesized at a slower rate than α
28
Q

RNA processing: Polyadenylation

(4)

A
  1. Termination: after AAUAAA sequence is recognized by enzyme complex, pol II continues txn for only about 30-40 nucleotides longer
  2. Endonuclease in enzyme complex cleaves sequence
  3. polyadenylate polymerase uses ATP to add ~200 A nucleotides
  4. Poly A binding protein associates to tail and mRNA tail wraps around
    * Protects from exonucleases
29
Q

RNA processing variation

A
  1. Mutations can lead to different splicing patterns (ß-thalosemia)
  2. PolyA sites recognized by enzyme complex can be dependent on cell type
  3. Alternate splicing; various splice sites
    * one primary transcript can make calcitonin in thyroid and CGRP in brain
30
Q

Human Papilloma Virus (HPV)

A
  1. Initial hyperplasia: virus not yet integrated into genome
    * mRNA has a short half-life
  2. Tumors: virus integrated into genome
    * increases txn; longer half-life
31
Q

tRNA processing

A
  1. Removal of 5’ and 3’ ends of mRNA by RNase P and RNase D
  2. tRNA nucleotidyl transferase adds CCA to 3’ end
  3. base modification
  4. splicing of internal portion
32
Q

tRNA structure

cloverleaf → t and D arm interact → folds to L shape

MHG 1 (10 decks)

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