Quiz 3 (Lec 8-9) Flashcards
(120 cards)
1
Q
central dogma of molecular biology
A
DNA –> RNA –> protein
2
Q
prokaryotic vs eukaryotic RNA polymerase similarieties
A
- overall structure quite similar
- 3 stage synthesis: initiation, elongation, termination
3
Q
RNA polymerase functions
A
1) recognize initiation sites and promoters
2) helicase activity: unwinds dsDNA
3) correct ribonucleotide triphosphate selection: unidirectional/processive
4) termination
5) activation/repression: transcription factors
6) fundamental reaction: 3’ OH attacks alpha P to create phosphodiester bond
4
Q
cis-acting elements
A
cis = on template strand being transcribed
5
Q
RNA synthesis direction
A
5’ to 3’, DNA is read 3’ to 5’
6
Q
RNA polymerase vs DNA polymerase
A
1) no primer required
2) no proofreading capability (1 in 10^5 error rate vs 10^10): acceptable because of codon degeneracy
3) slower (50nt/s vs 800)
7
Q
E. Coli RNAP structure
A
- 400kDA
- 4 subunits
- holoenzyme is two alpha, one beta, one beta prime, one sigma (pentamer)
- alpha2betabeta = core enzyme, contains catalytic site
8
Q
RNAP sigma subunit function
A
- decreases DNA binding affinity
- released after 10nts synthesized, goes to bind another core enzyme
9
Q
RNAP active site
A
- similar to DNAP
- Asp residues interact with DNA and Mg2+
- another Mg2+ cofactor interacts with ribonucleoside triphosphate gamma and beta phosphate = good LG
10
Q
footprinting
A
1) PCR amplification with radiolabeled DNA
2) add DNase I: cleaves randomly
3) denature dsDNA
4) gel electrophoresis
5) compare DNA/protein complex to naked DNA: gaps show where protein binds (DNase cannot access)
6) can use single base analysis to find sequence
11
Q
identification of prokaryotic promoter sites
A
- footprinting
- comparisons between different genes reveals similar initiation sites
12
Q
prokaryotic promoter sites
A
core promoter = 40nts, contains:
1) -35 region
2) -10 TATA (pribnow) box
13
Q
consensus sequence
A
- average of different sequences of genes
- number below shows percent frequency
14
Q
gene nucleotide numbering
A
+1 = initiation site
negative = upstream
positive = downstream
15
Q
RNAP interaction with promoter
A
- recognition helix in sigma subunit makes transient bonds with T and A
- residues: Tyr, Trp, Thr, Gln, Arg
16
Q
non-template strand
A
coding or sense strand
17
Q
template strand
A
non-coding or antisense strend
18
Q
RNA transcript resembles…
A
non-template strand with U instead of T
19
Q
strong vs weak promoters
A
- strong = frequently transcribed (housekeeping genes), 17nt between -10/-35 is ideal
- weak = multiple substitutions in -10/-35 regions, less frequently transcribed
20
Q
factors that impact promoter strength
A
1) regulatory proteins: bind near or to promoter regions
2) UP elements
21
Q
UP (upstream) elements
A
- bound by alpha subunit C-terminus
- increases transcription efficiency
- not highly conserved
- typically A/T rich
- 40-60 nts upstream
22
Q
alternative promoter sequences in E. coli
A
- bound by different variants of sigma subunit, ex:
1) standard = 70
2) heat-shock = 32
3) nitrogen-starvation = 54 - bacteria upregulates synthesis of different subunits in response to environmental changes
23
Q
prokaryotic transcription initiation mechanism
A
1) holoenzyme (sigma recognition helix) forms transient H-bonds with base pairs: rapid (10^10 M^-1 s^-1) and random search
2) recognition of promoter = closed promoter complex (STILL REVERSIBLE)
3) unwinding of DNA to form open promoter complex (IRREVERSIBLE)
4) RNA polymerase starts transcription, usually by adding a purine
5) sigma subunit falls off after 2-10 nts synthesized
6) core enzyme remains to continue to elongation
24
Q
open promoter complex characteristics
A
- 17bp segment = 1.6 DNA turns
- lower G-C content is easier to unwind
25
prokaryotic transcription elongation mechanism
1) formation of transcription bubble
2) nucleotide addition cycle: events required for addition of a nucleotide to the RNA product to form a cyclic process
26
prokarytotic transcription bubble
- RNA polymerase unwinds DNA at the front, ssDNA enters active site
- rewinds DNA at the back
- RNA-DNA helix formed as RNA synthesis occurs with 3' end of RNA in active site
- Phe in exit channel inserts into RNA-DNA helix to separate them, promoting exit of RNA strand
27
RNA-DNA helix characteristics
- ~8bps long = 1 double-helix turn
- moves 170 angstroms/sec = 50nt/sec
- rotates because of re/unwinding
28
nucleotide addition cycle important structures
- bridge helix
- trigger loop
- different conformations facilitate translocation and NAC
29
pre-insertion site in RNAP
- inactive site
- revealed by streptolydigin: inhibits transcription by sitting in insertion site, but RNAP could still bind NTPs
30
NAC steps
catalysis:
1) open trigger loop accepts NTP into pre-insertion site
2) closure of trigger loop moves NTP into insertion site
3) catalytic incorporation releases pyrophosphate
translocation equilibrium:
4) trigger loop opens
5) equilibrium formed between open with pre-translocated DNA, wedged with intermediate and open with post-translated DNA states
6) latter conformation allows another NTP to be accepted
31
general steps of transcription termination
1) ceasing of phosphodiester bond formation
2) RNA/DNA hybrid dissociation
3) rewinding of dissociated DNA strands
4) release of DNA by RNAP
32
two methods of termination in prokaryotes
1) intrinsic
2) protein-dependent
33
intrinsic termination signal in prokaryotes
- palindromic GC region separated by ~4A (forms U loop)
- followed by 4+ U (coded for by DNA)
- forms stem loop structure = RNAP stalls
- A/U rich area is weaker, leads to RNAP falling off
34
protein-dependent termination signal in prokaryotes
- Rho factor recognizes Rho termination sites
- used in rRNA synthesis: DNA template contains different Rho sites for 10S, 13S, 17S rRNA
- experiment: depending on timing of Rho addition = different subunits
35
how does Rho cause termination?
- binds to C-rich site: Rho utilization or rut site on synthesized RNA
- catches up to RNAP, causes unwinding of RNA/DNA helix = stalling + dissociation
36
Rho characteristics
- ATP-dependent helicase
- hexamer
37
drugs targeting transcription
1) rifampicin
2) actinomycin
3) amanitin
38
rifampicin action
- binds RND/DNA helix channel in prokaryotes (not eukaryotes)
- stops initiation
- no effect once elongation starts
39
actinomycin action
- intercalates with DNA
- DNA is no longer an effective template
- also affects DNA polymerase (replication)
40
steady-state level vs synthetic capacity of RNA
- synthetic capacity: cell's ability to synthesize
- usually correlated, except in the case of mRNA: immediately processed and transcribed
41
prokaryotic post-transcriptional modification of rRNA and tRNA
- usually on one transcript
- undergoes cleaving, processing (ex. CCA to tRNA), and modification (to bases)
- other mRNA typically undergoes little to no modification after synthesis
42
rRNA/tRNA mRNA cleavage
1) RNase III: cuts out rRNA
2) M16, 5, 23 (specific varieties): trims rRNA
3) RNase P: 5' end of tRNA
4) RNase D: 3' end of tRNA
43
mRNA base modification examples in prokaryotes
- ribothymidylate, pseudouridylate
- methylation ex. 6-dimethyladenine
44
prokaryotic vs eukaryotic transcription
1) spatial-temporal regulation: translation co-transcriptionally vs. must be exported out of nucleus (after fully processed)
2) processing: minimal vs extensive
3) number of RNAP: 1 vs 3
4) RNA subunits: 5 vs 7-10
45
characteristics of eukaryotic RNAP
1) all large proteins with 8-15 subunits
2) RNAP II has unique CTD with repeats of YSPTSPS
3) phosphorylation of CTD (S/T) regulates activity
4) different responses to alpha amanitin
46
eukaryotic RNAP I
1) location: nucleolus
2) cellular transcripts: 18S, 5.8S, 28S rRNA (one copy / primary transcript)
3) alpha amanitin effects: insensitive
4) promoters: A-rich upstream promoter element (UPE), ribosomal initiation element
47
eukaryotic RNAP II
1) location: nucleoplasm
2) cellular transcripts: mRNA precursors and snRNA (splicing)
3) alpha amanitin effects: strong inhibition, Kd = 10nm
4) promoters: TATA box, initiator element, downstream promoter element
48
eukaryotic RNAPIII
1) location: nucleoplasm
2) cellular transcripts: tRNA, 5S rRNA
3) alpha amanitin effects: inhibited by high conc., Kd = 1um
4) promoters: downstream promoters
49
important RNAPII subunits
- RBP1 contains CTD, homolog to beta prime subunit
- RPB4: promoter recognition
50
alpha-amanitin mechanism
- inhibits open to closed trigger loop change: ribonucleotide cannot move to insertion site
- inhibits wedged loop to open: RNAP cannot accept ribonucleotide
51
RNAP I promoter
core promoter:
1) -200 to -150: UPE
2) +1 ribosomal initiation element
52
RNAP II promoter
1) enhancer at least -1kB
core promoter:
2) upstream TATA box -100 to -20 OR DPE around +30
3) ribosomal initiation element +1
53
RNAP III promoter
type 1: 5srRNA
1) downstream A and C block
type 2: tRNA
1) downstream A and B block
*A/B/C all ~11bp
54
TATA box mutations
- single base markedly impairs promoter activity
55
upstream enhancers in RNAPII promoter
1) CAAT box
2) GC box
- usually for housekeeping genes
- increase transcriptional frequency through RPB3 binding
56
DNA looping
- binding of transcription activators to enhancer or silencer
- interaction with RNAP facilitated by DNA looping (to bring elements close together)
57
eukaryotic transcription initiation
1) TFIID (multi-subunit complex) recognizes TATA box via TATA box binding protein (TBP)
2) TFIIA stabilizes complex
3) TFIIF recruits RNAPII
4) TFIIB, TFIIE, TFIIH also recruited = basal transcription apparatus (BTA) aka pre-initiation complex
5) TFIIH (helicase) unwinds DNA and phosphorylates CTD
6) all except TFIIF dissociate
7) elongation begins: NAC
58
TFIID subunits
1) TBP
2) TAFs: recognize non-TATA elements, histone acetyltransferase activity
59
TFIIB function
recruits RNAPII and TFIIF, helps start-site selection
60
TFIIF function
- promoter targeting of RNAPII
- destabilizes nonspecific RNAPII-DNA interactions
61
TFIIE function
- recruited by RNAPII
- recruits and modulates TFIIH helicase, ATPase, kinase activities
62
TFIIH
- helicase activity
- CTD phosphorylation via 2 cyclin:CDK pair subunits
63
negatively regulated gene
- transcription prevented by a repressor
- transcribed in absence of active repressor
64
positively regulated gene
- transcription occurs in presence of activator
- not transcribed in absence
65
repressors and activators characteristics
- allosteric proteins
- bind ligands
66
positive transcriptional regulation methods
1) molecular signal causes binding of activator = transcription
2) molecular signal causes dissociation of activator = no transcription
67
negative transcriptional regulation methods
1) molecular signal causes dissociation of regulatory protein = transcription
2) molecular signal causes binding of regular protein = no transcription
68
operons
linearly organized:
regulator gene:
1) promoter for regulator gene
2) regulator gene
control sites:
3) promoter
4) operator (surrounding operon)
structural genes:
5) ex. lactose operon: z, y, a
69
polycistronic
mRNA that encodes multiple proteins
70
lac operon components and functions
1) z = beta-galactosidase: lactose --> allolactose
2) y = permease: transports lactose in
3) a = transacetylase: acetylates lactose to be exported out
71
lactose vs allolactose
1-4 beta-glycosidic linkage vs 1-6
72
lac operon two control mechanisms
1) regulatory response to lactose
2) regulatory response to glucose
73
lac operon lactose regulation
1) lac repressor prevents transcription
2) allolactose inactivates repressor and allows transcription
74
lac repressor mechanism
1) binds DNA (operator regions) as dimer
2) dimers from both operator regions forms tetramer = DNA looping
3) blocks RNAP
75
lac repressor transient binding
- transient binding ensures transcription occurs at least once = basal levels of lac operon genes
76
lac repressor/DNA interactions
- lac repressor alpha helix binds major groove
- H-bonding with AAs ex. Arg
77
lac repressor bound vs unbound
- lactose = unbound by allolactose = bound to DNA = very ordered
+ lactose = bound by allolactose = unbound to DNA = more disordered (conformational change)
78
results of lactose lac operon study
- adding lactose = beta-galactosidase and total bacterial protein (indicating growth) have direct relationship
- removing lactose = plateau
79
accessory proteins for promoters
some require additional accessory proteins to speed up transcription, ex, catabolite activator protein (CAP) for lac operon
80
CAP characteristics
- dimer of 22.5 kD peptides
- N-terminus binds cAMP
- C-terminus binds DNA
81
lac operon glucose regulation
1) increase in glucose = decrease in cAMP and vice versa
2) if glucose decreases, cAMP increase and binds CAP
3) CAP can bind to DNA: upstream of RNAP binding site (-41, -61 or -71 bp)
4) assists formation of closed promoter complex
82
purpose of catabolite repression
- ensures that operons for metabolism of alternative energy sources are repressed until glucose exhausted
83
components of eukaryotic transcription machinery
1) activators
2) repressors
3) coactivators
4) basal factors
84
common DNA sequences bound by proteins
- two-fold axis of symmetry
- palindromic
- dimer binding
- Glu/Asn often AT
- Arg often CG
85
DNA-binding motifs
80% DNA-binding proteins have one of:
1) helix-turn-helix motif
2) zinc-finger
3) leucine zipper-basic region (bZIP)
86
helix-turn helix
- recognition helix binds major groove
87
zinc fingers
- 10x small domains with Zn coordination to 4 Cys or 2Cys/2His
- key helix that binds DNA
88
leucine zippers
1) DNA binding region
2) 6-AA connector
3) leucine zipper: coiled coils held together by hydrophobic interactions between leucines (every 7th residue)
89
estrogen: example of hormonal control of transcription in humans
- cholesterol-derived hormone required for development and ovarian cycle
- diffuses across cell membrane
- ligand for DNA-binding protein (estrogen receptor)
90
estrogen receptor
- contain Zn fingers
- bind at estrogen-receptor elements
- three domains: transcription activation (variable), DNA binding (conserved, 66-68 residues), hormone binding (variable
91
estrogen receptor conformational change
- binding of estrogen causes helix 12 to fold up into side of receptor
- does NOT alter DNA binding affinity of receptor
- increases affinity of COACTIVATOR: P160 family = chromatin remodelling = increases transcription
92
drugs targeting estrogen receptor
- ex. tamoxifen
- binds in ligand-binding pocket
- prevents proper folding of helix 12, coactivator cannot bind
- ER antagonist to treat ER+ breast cancers
93
types of cellular RNA
1) mRNA: template for protein synthesis
2) tRNA: carries AAs to site of protein synthesis on ribosome
3) rRNA: part of ribosome
4) other: components of ribonucleoproteins with variety of functions
94
rRNA splicing
1) RNAPI produces pre-RNA with 3 ribosomal RNAs: 18S, 5.8S, 28S, separated by spacer regions
2) nucleotide modification: methyl groups and pseudouridines
3) spacer regions cleaved out
95
rRNA splicing machinery
- rDNA with many branches of pre-rRNA
- SSU (small subunit) processome (aka large ribonucleoprotein): at the ends for processing premRNA
96
tRNA splicing
1) RNAPIII produces tRNAs
2) 5' cleavage by RNAase P
3) 3' cleavage by RNase D
4) nucleotide modification
5) tRNA nucleotidyl transferase adds CCA to 3' end, releasing 3 PPi = AA attachment site
6) eukaryotes only: intron splicing near anticodon
97
eukaryotic mRNA delivery to ribosomes
- must be processed into mature mRNAs
- must be transported from nucleus into cytosol
98
eukaryotic vs prokaryotic mRNAs
- eukaryotic are monocistronic
99
mRNA capping
- 7-methyl-G capped added to increase transcript stability and define start site for protein synthesis
- occurs co-transcriptionally
100
mRNA capping steps
1) RNAP II produces mRNA
2) GTP added by guanylyl transferase:
a) hydrolysis of terminal phosphate on 5' end of transcript
b) diphosphate attacks alpha phosphate of GTP, PPi released = 5'-5' triphosphate linkage
3) methylation by RNA methyltransferase using S-adenosyl methionine:
a) cap O: guanine methylated at position 7
b) cap 1: 2'OH of sugar
c) cap 2: 2'OH of sugar
101
mRNA 3' polyadenylation steps
1) RNAP transcribes past consensus AAUAAA sequence (polyA addition site)
2) 10-35 nts past, RNAP stalls, resulting in cleavage and polyadenylation specificity factor (CPSF) recruitment
3) CPSF binds consensus sequence and invariant GU in mRNA = looping
4) cleavage factors (CFs) cleave downstream of consensus sequence
5) CFs and 3' fragment dissociate
6) poly(A)-adenylation protein (PAP) recruited to add 100-200 As
7) CPSF dissociates
102
timing of polyadenylation
closely linked to transcription termination
103
RNA editing
- change in nt sequence not as a result of splicing
- deamination of C to U or A to I, changing coding possibilities in transcript
- introduces diversity from same gene transcript
104
apolipoprotein
fatty acid and steroid transport protein
105
apolipoprotein RNA editing
1) in liver: unedited mRNA translated to ApoB-100 = LDL, VLDL surface
2) in small intestine: enzymatic deamination of CAA to UAA = early stop codon = no LDL receptor binding part = chylomicron surface
106
methods of increasing protein diversity by RNA editing
1) altering amino acid coding possibilities
2) introducing premature stop
3) changing splice site in transcript
107
organization of split eukaryotic genes
DNA:
1) promoter/enhancers
2) start of mRNA
3) exons and introns
4) poly-A addition signal
RNA:
1) 5' UTR
2) exons and introns
3) 3' UTR
mature mRNA
1) 7-mG cap
2) exons: as little as ~1/3 of DNA coding region!
3) 3'UTR with poly-A tail
108
alternative splicing
- more diversity of EXONs
- 2^n possibilities, where n is the number of exons that can be alternatively spliced
109
RNA sequences in intron for splicing
1) 5' splice site: invariant GU
2) branch site: key A residue
3) pyrimidine tract: ~10nts
4) 3' splice site: invariant AG
110
transesterification reaction
- exchange of ester groups
- requires no energy
111
two transesterification reactions in splicing
1) 2'OH on key A in branch site attacks phosphate of 5' splice site = precursor to lariat intermediate
2) 3'OH of exon attacks phosphate of exon 2 = spliced product + lariat form of intron
112
splicing branch point: lariat form of intron
adenine has 3 phosphodiester linkages:
1) 5' to 3'
2) 3' to 5'
4) 2' to 5' of guanine
113
snRNP
- small nuclear ribonucleoprotein particles
- required for splicing
- small RNA (100-200 bases) + ~10 proteins (some general and specific)
- regions that bind RNA are protein-deficient to facilitate base-pairing
- major snRNP species are abundant, >100 000 per nucleus
114
key snRNP in pre-mRNA splicing
1) U1: 5' splice site
2) U2: branch site
3) preformed complex of U4-6: 5' splice, recruitment of branch point to 5'splice site
115
splicing steps
1) U1 recognizes 5' splice site
2) ATP hydrolysis for U2 binding to branch site
3) ATP hydrolysis for U4-6 complex formation + ATP for binding
4) once U5 aligned at 5' splice site, ATP for U4 dissociation unmasks U6 activity = U2/U6 catalytic site forms across pre mRNA
5) U5 uses ATP to align 2’OH of the A branch site and 5’ intron splice site for first transesterification reaction
6) U5 uses ATP to align3’OH of exon 1 with the 3’ intron splice site for second transesterification
7) ATP releases U5/6/2, another to dissociate U2/6
116
spliceosome catalytic center
- U6/2 base pair
- U2 base pairs with branch site = key A residue protrudes
- dissociated by ATP dependent helicase
117
CTD recruits which proteins to pre-mRNA
1) capping enzymes
2) splicing components
3) endonuclease
118
how does CTD recruit proteins?
phosphorylation
119
CTD coupling transcription to premRNA processing mechanism
1) phosphorylated CTD has capping enzymes, splicing factors and polyadenylation factors (starting from end inwards)
2) capping
3) spliceosome recruited, splicing factors splice
4) poly(A) added
120
issue of complex RNA processing
difficult to trace mature mRNA back to original gene sequence
