Exam 3 Flashcards
(121 cards)
1
Q
Central Dogma of Molecular Biology
A
DNA –> RNA –> RNA processing + translation –> protein
2
Q
Gene
A
-all information needed to synthesize RNA/protein
-not all genes encode proteins
3
Q
Open reading frame (ORF)
A
-protein coding sequence
-long >80 string of amino acid-specifying codons that begin with AUG codon and is not interrupted by stop codons
-found after promoter on operon/gene
4
Q
Promoter
A
-location on DNA where RNA polymerase binds
-determins where transcription starts (-10, -35 regions)
5
Q
Operon
A
-two or more adjacent genes that are coordinately controlled by transcription from a single promoter
-single mRNA that carries multiple protein coding sequences
-read in 3’ –> 5’ direction
-prokaryotic genes often organized in operons
-no introns
-multiple ORFs present in one transcription
6
Q
Prokaryotic gene expression
A
-circular chromosome
-no nucleus
-DNA replication, transcription, and translation occur in same cellular compartment
7
Q
DNA vs RNA synthesis
A
-DNA synthesis: takes place in nucleus and mitochondria, monomer substrates are dNTPs, next nucleotide added at 3’OH, both strands of DNA are template, DNA polymerase (needs primer), synthesis begins are origin of replication
-RNA synthesis: takes palce in nucleus/cytoplasm, monomer substrates are rNTPs, next nucleotide added at 3’OH, one strand of DNA is template, RNA polymerase
8
Q
RNA synthesis
A
-analogous to DNA synthsis
-synthesis is 5’ to 3’, nucleotides added to 3’OH of previous nucleotide
9
Q
Transcription
A
-the process of copying a segment of DNA into RNA for the purpose of gene expression
-DNA is template (non-template = coding strand)
-RNA polymerase binds to DNA at promoter
-helix is unwound to form transcription bubble (rewinds after transcribed)
-RNA is synthesized in 5’ to 3’ direction with rNTPs
-5’ end is displaced from template as polymerase moves
10
Q
messenger RNA (mRNA)
A
-encodes proteins
-created during transcription by RNA polymerase
-is read by a ribosome in the process of synthesizing a protein
11
Q
transfer RNA (tRNA)
A
-RNA adaptor molecule that helps decode mRNA sequence into a protein
-carry a specific amino acid
-has clover leaf structure
-anticodon loop matches to a specific mRNA codon
-amino acid attachment site is at 3’ (-OH) end
-inosine (I) is present in anticodon loop of some RNA molecules
12
Q
ribosomal RNA (rRNA)
A
-noncoding RNA that is main component of ribosomes
-ribozyme which carries out protein synthesis
13
Q
small RNA (sRNA)
A
-noncoding RNA
-regulates gene expression through gene silencing
14
Q
Three phases of transcription
A
-Initiation
-Elongation
-Termination
15
Q
Prokaryotic vs Eukaryotic RNA polymerase
A
-Prokaryotic: consists of 6 subunit A2BB’wσ holoenzyme responsible for initiation and 5 subunit A2BB’w responsible for elongation
-Eukaryotic: many subunits
16
Q
Initiation
A
-RNA polymerase recognizes and binds to promoter sequences, determines starting point for transcription (+1)
17
Q
Elongation
A
-RNA polymerase monitors binding of rNTP to next base on template
-If match, catalyzes bond formation
18
Q
Termination
A
-RNA polymerase pauses and dissociates from the template
-Usually at designated termination site
19
Q
Prokaryotic RNA polymerase subunit roles in initiation
A
-a: enzyme assembly, promote interaction with regulatory proteins
-B and B’: catalysis
-σ: positions holoenzyme to initiate trx, Binds to -10 and -35 regions of promotor, fits in major groove and binds with R-groups, dissociates from RNA pol after elongation begins
20
Q
Intrinsic mechanism of termination
A
-Termination sequence: G-C rich region followed by string of A’s
-After transcription through 3’ untranslated region (UTR) RNA forms hairpin loop
-Triggers release of completed transcript and RNA polymerase from DNA
21
Q
Rho-dependent mechanim of termination
A
-Rho (ρ) protein binds to rut site in 3’ end of the RNA
-Acts as a helicase to disrupt the RNA-DNA hybrid of a paused polymerase
-Facilitates dissociation of RNA from DNA template
22
Q
Regulation of gene expression
A
-Prokaryotes and other single-celled organisms respond to changes in their environment through altering gene expression
-Prokaryotes are very efficient: only express genes and produce enzymes when they are needed
-Couple expressions of genes to a sensing system
-In multicellular organisms, changes in gene expression allow for acclimation to different environmental conditions and for formation of different cell types (development)
23
Q
Basal expression in operon
A
-RNA polymerase binds occasionally to promoter
-RNA polymerase alone (no activator and no repressor)
-Expression at low (basal) level
24
Q
Negative regulation in operon
A
-binding of a repressor blocks binding of RNA pol
-Inhibits transcription
-inhibited transcription in absence of inducer (repressor binds to DNA at operator)
-basal transcription in presence of inducer (repressor does not bind to DNA at operator)
25
Positive regulation in operon
- binding of an activator recruits RNA pol
-Expression is activated
-basal transcription in absence of inducer (activator does not bind to activator binding site)
-high transcription in presence of inducer (activator binds to DNA)
26
Activator protein
-mediates positive regulation, bind to activator binding sites of DNA
27
Repressor protein
-mediates negative regulation, bind to operators of DNA
-Operators and activators are different DNA sequences, often located near the promoter
28
Inducer
-change the conformation of activators and repressors
-Inducer binding leads to activator binding to DNA
-Inducer binding leads to repressor not binding to DNA
-Two different inducer compounds
29
Polycistronic
A single mRNA that contains multiple protein coding sequences (such as operon)
30
Lac operon components
-LacZ: encodes B-galactosidase (lactose → glucose → galactose)
-LacY: encodes Permease (permits entry of lactose into cell)
-LacA: encodestransacetylase
-LacI: encodes Lac repressor
-LacP: promoter region
-LacO: operator region
31
Glucose in Lac operon
-Glucose (monosaccharide) is preferred by bacteria as an energy source (catabolite/simpler form of lactose)
-If both glucose and lactose are present, an extra layer of regulation ensures that bacteria does not invest energy in utilizing other sugars until easily digested glucose is exhausted
-Mechanism for sensing glucose relies on inverse correlation between glucose and cAMP levels; presence of glucose leads to down-regulation of adenyl cyclase enzyme activity
-CAP = cAMP receptor protein
-High glucose = low cAMP
-Low glucose = high cAMP
32
Lactose in lac operon
-Lactose is both inducer and substrate (for LacZ)
-Even in absence of lactose, sometimes repressor will not be bound to operator and there will be low level of transcription of lac operon
33
Positive control of lac operon
-activation of lac operon in the presence of lactose (inducer) but low glucose (high cAMP)
-cAMP levels are high, CAP w/ 4 cAMP binds to CAP site, facilitating binding of RNA polymerase to promoter
-High transcription levels
34
Negative control of lac operon
-repression of lac operon in absence of lactose (inducer)
-Lac repressor protein binds to LacO and prevents RNA polymerase from binding to promoter
-Inhibits transcription of lac operon
35
Partial diploid
-normal chromosome plus an F’ plasmid with 2nd copy of small DNA segment (ex. Add 2nd copy of lacl gene)
-Ask if relative position of alleles is important
36
cis-acting elements of operon
-sequences that can only affect other elements on the same DNA strand it is on
ex. LacP and LacO (cannot restore expression on chromosome from plasmid location)
37
trans-acting elements of operon
-proteins/molecules encoded by separate genes that can diffuse to other DNA molecules and regulate genes on those molecules
-ex. LacI (can affect lac operon from F' Plasmid)
38
cis and trans acting factors of genes in euakryotic and prokaryotic cells
-Regulatory proteins can be encoded by genes physically distant from those they regulate (proteins can diffuse in the cell or nucleus)
-In prokaryotes, usually directly adjacent to genes they regulate
-In eukaryotes, regulatory elements can function at a greater distance
39
B-form DNA structure
-Major groove: deep, wide, good size to fit a-helix of a DNA binding protein
-Minor groove: narrow shallow
-Lac repressor and many other bacterial DNA binding proteins bind to DNA using helix-turn-helix DNA binding domains
-lac repressor hydorgen binds to operator as dimer into major groove, causing DNA bending
40
operator
-regulatory DNA sequence that control the transcription initiation of a gene or group of genes
-usually binding site of repressor
41
RNA and protein linear code
-RNA linear code = 4 letters (A, U, C, G)
-Protein linear code = 20 letters (amino acids)
-triplet code: 4^3 = 64
42
Redundancy of genetic code
-there are only 20 amino acids, not 64 (like triplet code)
most amino acidsa re specified by two or more codons
-synonymous codons specify for the same amino acid
43
How genetic code is read
-Transfer RNA (tRNA) molecules
-aminoacyl-tRNA synthetases
-ribosomes
44
Wobble base pairing
-presence of inosine on anticodon allows tRNAs to recognize mutltiple codons ending in U, C, or A (3rd position by 5' end)
45
Amino-acyl tRNA synthetase
-enzyme responsible for linking specific amino acids to the tRNAs that recognize the codons for that specific amino acid
-Carboxyl group of amino acid is linked to 3’ OH end of tRNA
-Adaptor molecules brings amino acids corresponding to each codon to ribosome
-Amino acid attachment at 3’ end: protein language
-Base-pairing with mRNA at 5’ end: nucleotide language
46
Charging of tRNA to specific amino acids via amino-acyl tRNA synthetase
-amino acid binds to active site of specific amino-acyl tRNA synthetase
-tRNA binds to amino-acyl tRNA synthetase with amino acid, transfers to tRNA
47
ribosomes role in translation
-binds mRNA and TRNA-AminoAcid
-three tRNA binding sites: A, P, E
48
A site (ribosome)
-Acceptor
-first tRNA binding spot
-location where incoming aminoacyl-tRNA first attaches to ribosome
-30S subunit
49
P site (ribosome)
-peptidyl
-second tRNA binding spot
-peptide bond between the growing polypeptide chain and new amino acid is formed
-peptidyl transferase is enzye that catalyzes synthesis of peptide bonds between amino acids
50
E site (ribosome)
-Exit
-final (third) tRNA binding spot
-50S subunit
-exit for deacylate tRNAs (ones that have released their amino acids)
51
Ribosome composition
-two subunits
-large = 50S
-small = 30S
-both subunits contain both rRNA and proteins
-rRNA makes up most of ribosome in eukaryotic ribosomes
-proteins are stabilizers
52
Initiation of translation (prokaryotes)
-Initiation factor 2 (IF2) delivers special tRNA charged with fMet to P site (AUG)
-base pairing between 16S rRNA and Shine Dalgarno sequence (ribosome binding site on mRNA, around 8 bp from start codon AUG)
-establishes ORF
-once 30S subunit is in place, IF2 brings fMet
-once tRNA-fMet is in place, 50S su binds to 30S su
53
Elongation of translation
-Elongation factor EF-Tu (G protein in GTP bound form) forms complex with charged tRNA
-tRNA-AA (ternary complex) is loaded into A site or ribosome, if codon/antiocodon interacction is correct, GTP on EF-Tu is hydrolyzed and EF-Tu is released
-conformational change of A site occurs
-peptide bond is formed between fMet and amino acid on tRNA (between B on tRNA in A and C=O on tRNA in P)
-ribosome moves forward
54
fMet (formylmethionine)
-first amino acid inserted into polypeptide chain
-often removed after translation is complete
-initiator amino acid
55
Initiation factor 2
-delivers a special tRNA (initiator tRNA) charged with formylmethionine (fMet) to P site (usually AUG)
-establishes ORF
56
EF-Tu
-elongation factor thermo-unstable
-another G protein in its GTP-bound form) forms a complex with a charged tRNA
-delivers aminoacyl-tRNAs to ribosome (A site)
-ensures that the correct amino acid is added to growing polypeptide chain
-hydrolysis of GTP allows it to interact with tRNA (EF-Tu is released as EF-Tu with GDP)
-delivers fMet-tRNA to AUG codons within ORF during elongation
57
Ribosome as ribozyme
-active site of ribosome contains only RNA
-ribozyme: RNA molecules that exhibit enzymatic activity
-solely the RNA provides catalytic activity
58
Termination of translation (prokaryotes)
-Elongation cycle continues until a stop codon is encountered
-Release factors (RF): protein that mediates termination by interacting with stop codons in A site (Structurally resembles tRNA)
-Ribosome dissociates, tRNAs and completed polypeptides are released
-Molecular mimicry (release factor is protein that mimics structure of tRNA, allowing them to bind)
59
Release factors (RF)
-protein that mediates termination by interacting with stop codons in A site
-Structurally resembles tRNA
60
Molecular mimicry (translation termination)
release factor is protein that mimics structure of tRNA, allowing them to bind
61
Polysome
multiple ribosomes translating a single mRNA transcript
62
Gene expression significance in eukaryotes
-Changes in single-celled organisms (prokaryotes and unicellular eukaryotes) allow the organism to adapt to different environmental conditions
-Changes in multi-cellular eukaryoteic organisms allow
formation of different cell types and adaptation to different environmental conditions
63
Features of transcription in eukaryotes
1. Spatial separation of transcription and translation in eukaryotes
2. More complex transcriptional regulation in eukaryotes (3 RNA polymerases, General transcription factors (GTFs), Cis-acting elements (DNA regulatory sequences that bind proteins) are more varied and can be positioned in different configurations relative to the coding sequence)
3. Extensive processing of primary transcript destined to become (mature) mRNA in eukaryotes
64
Nucleosomal packaing of DNA in eukaryotes
DNA double helix → nucleosome → chromatin → scaffold-associated chromatin → condensed heterochromatin → compacted chromosome
65
Chromatin
-Eukaryotic DNA is packaged into a protein-DNA complex wrapped around histone
-Composed of nucleosomes
-makes up chromosomes
66
Nucleosome
-section of DNA that is wrapped around a core of proteins (histones)
-basic building block of chromatin
-DNA is wrapped around a protein core made of 8 histone molecules (H3(2)H4(2)H2A(2)H2B(2) + H1 (linker))
67
Histone
-proteins that package DNA into structural units, nucleosomes, which are fundamental building blocks of chromatin
-bind to DNA, help give chromosomes their shape, and help control the activity of genes
-4 different histone proteins form a histone octomer (H3, H4, H2A, H2B x 2 and a single H1 linker)
-interact with DNA minor groove
-amino terminal tails provide binding site for proteins to covalently modify chromatin structure (through acetylation and methylation)
68
Writers
-proteins that covalently modify histone amino acids
-ex. Methyl transferases, Acetyl transferases (HATs)
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Erasers
-proteins that restore modified histone amino acids to unmodified form
-ex. Demethylases, Deacetylases (HDACs)
70
Readers
-proteins that bind to modified histone amino acids
-ex. Methyl readers, Acetyl readers
71
Mammalian gene structure
-promoter sequence adjacent to transcription start point determines where transcription starts
-consist of core promoter inside promoter and an enhancer
72
Core promoter (mammalian genes)
-set of sequences required to recruit General Transcription factors and RNA polymerase to non-chromatin DNA in a test tube is called the promoter
-contains binding site for GTFs (adjacent to transcription start site and required for RNA polymerase binding to promoter)
73
Promoter (mammalian cells)
-core promoter plus nearby elements
-minimal sequence required to recruit RNA polymerase to transcription start site in a cell (to DNA packaged into chromatin)
74
Enhancer (mammalian cells)
-work from far away to increase transcription; they are needed for maximal promoter activity
-Often involved in cell-type specific expression (ex. Liver, muscle, gut)
-Many human genes have multiple enhancers, spread over large distances, each of which is responsible for activating expression of the gene at a specific time/place
-Example: sonic hedgehog (Shh) gene is a developmental regulator that has enhancers spread over more than a megabase of DNA
75
Bacterial vs Eukaryotic regulation
-Bacterial: ground state is on, template for transcription is protein-free, activators enhance weak polymerase binding, repressors interfere with polymerase binding, polymerase holoenzyme binds protein-free DNA, promoters are DNA
-Eukaryotic: ground state is off, template for transcription is chromatin which is inaccessible to RNA polymerase, activators make chromatin accessible and/or promote the formation of a marked site on DNA, repressors interfere with transcription by blocking activators.making chromatin less accessible, RNA polymerase binds to protein DNA-complexes which mark DNA, promoters are really protein-DNA complexes embedded in a chromatin environment
76
GTF (growth transcription factors)
-molecules that regulate gene expression and play role in growth and development
-In absence of chromatin, eukaryotes require GTFs for transcriptional machinery
-GTFs and RNA polymerase assemble at the core promoter to form a Pre-initiation complex (PIC), which is required for basal transcription in a test tube
77
TFIIB
-transcription factor II B
-general transcription factor crucial for RNA pol II transcription initiation
-responsible for start site selection
-stabilizes TBP-TATA complex
-recruits pol II/TFIIH
-made of p33
77
General transcription factor
-essential proteins that facilitate the initiation of transcription by RNA polymerase
-bind to specific sites (promoter) on DNA and help assemble PIC (total molecular weight: 1 megadalton)
-includes TFIIB, TFIID, and TFIIH
78
TFIIH
-transcription factor II H
-helps RNA pol II bind to promoter and initiation transcription
-TFIIH helicase catalyzes DNA strand separation
-TFIIH kinase phosphorylates carboxyl terminal domain (CTD) of RNA pol II
-also participates in transcription-coupled nucleotide excision repair
79
TFIID
-transcription factor II D
-large multi-subunit complex (TATA binding protein and 14 associated factors) that recognizes core promoter DNA and helps assemble PIC
-binds to TATA box and initiator on core promoter
-TBP acts as anchor for PIC
80
RNA Pol II (translation)
-responsible for transcription initiation/elongation/termination, recruitment of mRNA capping enzymes, CTD phosphorylation
-binds to TFIIB at promoter
81
RNA pol II pre-initiation complex (PIC)
-large multiprotein assembly required for euakryotic transcription initiation, formed at promoter region of gene
-sequential recruitment of general transcription factors leads to assembly of PIC:
1. TATA box binding protein: part of TFIID, acts as anchor
2. TFIIB
3. RNA pol II (binds to TFIIB)
(Does not bind directly to core promoter)
4. TFIIH
82
C-terminal domain of RNA pol II
-unphosphorylated carboxyl-terminal domain (of largest subunit) is a promoter tether
-phosphorylated by TFIIH kinase (opens chromatin), which initiates transcription and allows RNA pol II to escape promoter and produce mRNA
-an “integrator”/”Coordinator” of the gene expression machinery during elongation
83
Histone lysine acetylation
(how to make chromatin available for GTFs and RNA pol)
-weakens DNA binding (eliminates charge difference)
-Histones (unlike acidic DNA with negatively charged phosphate) is basic due to positively charged lysine and arginine amino acid residues
-Lysines in amino terminal tails can be modified by acetylation, which neutralizes lysine’s positive charge
-Loosens chromatin structure by relieving nuelcosome interactions and histone-DNA interactions
-Provides binding sites for proteins that facilitate activation (readers)
-Step in gene activation
84
Histone modifications at promoter
-regulate eukaryote transcription
-histone acetylation (loosens chromosomes, high transcriptional activity)
-ground state chromatin (inaccessible to transcription machinery, transcriptional activity is turned off)
-histone (H3K9) methylation (further condenses chromatin (heterochromatin), transcriptional activity is turned off (low))
85
Elements required for transcription when DNA is part of chromatin
-1. Chromatin remodeler: to reposition nucleosomes (ATP-depenent)
-2. Formation of Transcription Initiation Complex:
-3. Complexes that mediate these interactions (and long range-enhancer/promoter interactions)
86
Chromatin remodelers
-use energy of ATP hydrolysis to repositon/restructure nucleosomes
-Nucleosome remodeling is step in gene activation
-ATP dependent chromatin remodeling complex binds to nucleosome and ATP → ADP causes nucleosome sliding
87
Mediator
-not required for basal transcription in vitro but is considered to be a GTF, required for transcription of most genes in cells (chromatin context)
-interacts with RNA polymerase/GTFs/other activator proteins bound to far away enhancers
88
Activators and repressors transcription
-bind to enhancers/nearby elements
sequence-specific transcription activator proteins activate transcription
-Activators have two seperable domains for DNA binding and activation
-If DNA binding domains are “swapped out” for one another, activation still works (interchangable)
-Targets of activation domains are recruited to promoter by protein-protein interactions among these targets (co-activators)
89
Coactivators
-proteins that increase rate of transcription for genes
-include General transcription factors (TFIID-major one (TBP with TAFs)), Mediator complex-associated with RNAP, Histone Code Writers
(HATs and Histone Methyltransferases), and Chromatin remodeling complexes
90
Transcription regulation via nutrient availability in yeast
-GAl7, GAl10, GAL1 genes in yeast are coordinately regulated by a transcriptional activator, Gal4
-Gal4 proteins bind to yeast enhancers (upstream activation sequence, UAS) to activate transcription when galactose is present
-In absence of galactose, Gal80 protein binds to Gal4 activation domain
-While Gal4 still binds to DNA, it can no longer activate transcription
-In presence of galactose, galactose binds to Gal3 protein, which binds to Gal80 in the cytoplasm, preventing it from getting to nucleus
-Gal4’s activation domain is now exposed to promote transcription initiation of gAL7, GAL10, and GAL1
-Gal4 activation domain binds a histone acetyltransferase coactivator complex (SAGA) and mediator, which recruit GTF’s and RNA polymerase to promoter
91
HAT (histone acetyl transferase) and HMT (histone methyl transferase)
-chromatin modifiers
-bind to phosphorylated RNAP CTD tail
-HAT (writer) activates chromatin as the polymerase transcribes (adds acetyl groups, makes DNA more loose/accessible)
-HMT (writer) methylates histone at H3K36, recruiting histone deacetylase (HDAC) (adds methyl group)
92
histone deacetylase (HDAC)
-erases the acetyl marks to "inactivate" the chromatin after the polymerase has passed through, recondensing chromatin
-inhibits transcription
-co-repressor (recruited by repressor MIG1 to inhibit GAL1 transcription)
93
E2F
-family of proteins crucial for regulating cell cycle progression
-act as activators or repressors, depending on association with RB
-binds at promoter of cell-cycle regulated genes
-growth factor (signal to divide) activated kinases phosphorylate RB at restriction point of cell cycle
94
RB
-co-repressor complex with HDAC activity or co-activator with HAT activity
-when not phosphorylated, EF2 associates with HDAC, resulting in repression of transcription of cell cycle regulated genes (repression of transcription)
-When RB is phosphorylated, EF2 associates with an HAT, resulting in activation of transcription of cell cycle regulated genes (activation of transcription)
95
heterochromatin
-long-range repressed chromatin
-found in constitutive (always present) form at telomeres and sequences that flank centromeres in many organisms
-densely packed, transcriptionally silent
-Heterochromatin establishment requires the participation of specific non-histone chromosomal proteins that associate with the “silenced” DNA
-Heterochromatin boundary (prevents spreading of heterochromatin) is maintained by activating competing histone modifiers (HATs and HMTs)
96
telomere
- sequence at the end of DNA that protect DNA and prevent chromsomes from fusing
-Shortens with aging
97
Centromere
-constricted region of chromosome that joins sister chromatids together
-attachment point for spindle fibers
98
HP1
-protein that binds to methylated histone tails (H3K9) and recruits histone methyltransferase (further silences genes)
-maintains heterochromatin
99
Methylation of H3K9
-lysine (K) at position 9 of the N-terminal tail of histone H3
-Marks repressed chromatin
-associated with heterochromatin and epigenetic silencing
100
Methylation of C5 of cytosines
-in cpG dinucleotides (dna sequence in which cytosine is followed by guanine, found near promoters)
-DNA methylation is established by DNA methyl transferase (DMNT), which puts a methyl (CH3) group at the 5 position of a cytosine if it is followed by a guanine (CpG sequence), forms 5MeC
-MBD proteins recognize methylated DNA and help maintain chromatin in an “off” state
1. 5MethylC binding domain (MBD) proteins recognize 5meC modified DNA
2. MBD proteins recruit histone deacetylases (HDACs)
3. De-acetylated chromatin is in an “off” state
101
Genetic vs Epigenetic alterations
-Genetics: DNA changed but transcription still occurs (ex. Mutation)
-Epigenetics: DNA is unchanged but transcription doesn’t occur (ex. Repressing chromatin structure)
102
DNA methylation
DNA methylation blocks binding of transcription factors to DNA, which prevents initiation of transcription (silences genes)
103
RNA processing in eukaryotic cells
-Steps:
1. 5’ mRNA capping
2. Cleavage and polyadenylation (poly A) (Addition to generate the 3 end of the mRNA)
3. Intron splicing (Removal of intron sequences)
-Occurs in the nucleus prior to export to cytoplasm
104
Transcription of mammalian pol II gene
Primary transcript (pre-mRNA) – capping, cleavage/polyA addition, splicing → processed transcript (mRNA)
105
5' mRNA capping
-1. Removal of the terminal Pi from the 5’ end
-2. A guanine cap is attached to the 5; end using GTP (releasing PPi)
5’-to-5’ linkage
-3. Methylation of guanine and sometimes ribose
-Function:
--Stall factor stops pol II shortly downstream of the initiation site
--Cap addition complex (capping enzymes) bind to phosphorylated CTD and adds the cap to the 5’ end of the mRNA
--Increase transcript stability by protecting against ribonucleases
--Increase translation efficiency (5’ cap binds to translation initiation factors)
106
Addition of polyA tail to 3' end of pre-mRNA
-Upon cleavage signal, new RNA is cleaved by specific endonuclease
-ATP donates P, inciting addition of tail by polyA polymerase (untemplated)
-Result is polyadenylated mRNA precursor (with polyA tail)
-Function:
--Stability of mRNA
--Enhancing translation efficiency
---Blocking of polyA synthesis does not interfere with synthesis of primary transcript (mRNA molecules with no polyA tail can be transported out of nucleus, but protein synthesis is less efficient)
107
Intron
-large segments of noncoding DNA that are removed before RNA translation
-Typically represent the majority of the sequence of the gene
108
Exon
small segments of coding DNA, are retained in mRNA trasncript
109
Transesterification reaction
-one phosphodiester bond is broken and another is made
-First transesterification occurs at branch (A) and 5’ splice site
--Phosphodiester linkage made at 2; -OH of branch point
-Second transesterification occurs at 3’ splice site and 5’ splice site
110
phosphodiester bond
a covalent linkage between the phosphate of one nucleotide and the hydroxyl (OH) group attached to the 3′ carbon of the deoxyribose sugar in an adjacent nucleotide
111
Spliceosome
-mediates reactions in intron splicing, removes introns via splicing
-Composed of small nuclear ribonucleoproteins (snRNPs)
112
Intron splicing
-two steps of transesterification reactions
-Splicing steps:
--Splicing begins with recognition of the 5’ splice site by the 165 base long U1 snRNA found in the U1 snRNP
--SNPs U1 and U2 bind to the 5’ splice site and internal A
--U4-U5-U6 complex joins the spliceosome, resulting in folding
--First splicing reaction occurs (one intron ends attaches to branch point)
--Second splicing reaction occurs (other intron end is cleaved, exons join)
113
Alternative splicing patterns
-One gene can code for multiple (isoforms) depending on the specific intron splicing pattern
--More than 50% of human genes are subject to alternative splicing and/or RNA processing
--Example: Tropomyosin isoforms
-For an exon to be defined, it must have a good, recognizable 5’ splice site and 3’ splice site
-Splicing is in general exon defined
-Splicing is regulated by proteins binding to mRNA and spliceosomes
-Defects in alternative splicing are often the cause of genetic diseases and cancer cells
114
Splicing repression
RNA binding proteins bind exons or introns (at intronic/exonic splicing silencer sites (ISS and ESS) at/near consensus sequence elements and interfere with spliceosome assembly
115
Splicing activation
-RNA binding proteins bind to intronic/exonic splicing enhancer elements (ISE and ESE) and promote spliceosome assembly at intron/exon boundaries with “poor” 3’ or 5’ sequence elements
116
Activator-Dependent Inclusion (splicing)
-features a weak 5’ or 3’ splice site, which leads to the requirement of an SR activator binding to an exon splicing enhancer
-If activator is present, the exon is defined and included in the mRNA
-If the SR activator is absent, there is no U1 snRNP binding, the exon is not defined and is therefore excluded (exon looks like part of intron)
117
Repressor-dependent exclusion
-the 5’ and 3’ splice sites are “good” (have consensus sequence). The presence/absence of a repressor determines if an exon is defined
-If the repressor is present, the U1 snRNP and/or the U2 snRNP do not bind
--The exon is not defined and is excluded (exon looks like intron)
-If repressor is absent, the exon is defined and is included in the final mRNA
118
Self-splicing introns
-the intron RNA folds into a structure that catalyzes its own removal, RNA functions as an enzymes (ribozyme)
-Involves twotransesterification reactions
-3’-OH of a guanine nucleoside attacks the 5’ phosphate at the 5’ splice site of the mRNA
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Alternative splicing in humans (Calcitonin/CGRP)
-Calcitonin/CGRP gene
-Calcitonin: binds to GPCR, decreases blood calcium
-Calcitonin Gene Related Protein (CGRP): binds to GPCR, elevates cAMP in target cells
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GPCR
-G-protein coupled receptor
-proteins that act as signal receptors, transmit signals from outside to inside of cell
