Exam 3 Flashcards
RNA Splicing: An Introduction
Exon:
Any RNA seq coding or non that is retained in the mature mRNA (RNA ready to be translated)
Intron:
Any RNA seq that is removed from the mature mRNA
Who’s got what:
No introns: Prokaryotes (Some do have self splicing) and viruses Introns and Exons: Eukaryotes but do have some with few or no introns.
Interesting Facts about Introns
Average # per gene:
Seems to increase with increasing “organism Complexity”
Size:
In general introns are much larger than exons
Portion of primary RNA:
-Range of 150 nt in an intron to 800,000
-Usually the largest portion of pre-spliced mRNA
Extreme Example:
- mammalian dihydrofolate reductase: Pre-mRNA is 31,000 nt (31 kb) and spliced is 2,400 nt
-human dystrophin: Gene is 2,400,000 nt long, RNAP sythnesis RNA at – 40 nt/sec takes 17 hrs to transcribe gene
Evolutionary Implications of RNA Splicing
One Theory:
Introns evolved from transposons splicing evolved to some transposon insertion
Another Theory:
Introns have always existed prokaryotes originally had them and lost them
Exon Shuffling Theory:
Introns provide cells a buffer zone to recombine exons as a single unit into new genes
Evolutionary Advantage:
Introns provide us a buffer zone for mutations
Intron Splicing Classes
3 Mechanisms (Classes):
Spliceosome-mediated splicing: most common almost all euk genes use this pathway
Self-splicing
An intron that folds into an active structure and splices itself out
Group II Introns and Group I Introns: found in a few prokaryote and organelle genes
What is transcription?
Formal Definition:
Synthesis of RNA from a template
How it occurs
DNA-dependent RNA synthesis: “normal” tscript
RNA-dependent RNA synthesis: Synth of DNA from RNA template
Reverse transcription:
Synth of DNA from RNA template (not really tscript)
RNA Polymerase: The Claw
Active site at base pinchers
General Info: contains 4 subunits:
β, β’ (create active site)
α2 and ω (proteins bound to β, β’)
General Structure:
Pincers: β, β’ interacting Active Site Cleft: formed when β, β’ bind
Catalytic Mechanism: Requires 2 divalent actions to catalyze the new phospidester bond one cation leaves after each rxn
Main Steps of Bacterial Transcription
1) Initiation:
RNAP binds promoters upstream of transcriptional start site (+1)
2) Elongation:
RNA Elongates mRNA
3) Termination:
Tscript is terminated
defined as when RNAP leaves DNA (NOT when mRNA leaves RNAP)
Introducing DNA Consensus Sequences: When You’re Trying To Find a Pattern That Might Mean Something Biological
Sequence Alignment:
Consensus Sequence:
Bacterial Transcription: Initiation
1)RNAP binds promoters
Promotor elements bound by RNAP and/or tscript factors
Consensus Sequence:
1) -10 box (pribnow seq) (TATAAT) 2) -35 box (TTGACA) 3) 2 reigons are highly conserved for many e.coli genes
Sigma Factors (σ70, σ32, σ54…)
General Information: E.coli and another bacteria evolved a 5th subunit: Sigma to RNAP
DNA Binding:
- binds -35 with helix-turn-helix
-too complex to describe
-10 binding region is complex
Diversity:
E.colli has 7 sigma factors
σ70 - normal unstressed growth
σH, σ32- heat shock sigma factor
Bacterial Transcription: Initiation (continued)
Some Enzyme Mechanics:
Irreversible step: "melting" of the DNA reigon into single strands Abortive Initiations: RNAP starts many times synth 1-9 nt from +1 stops and returns to +1 Escaping the promoter: If you can scrunch at least 10 nt may develop enough tension tp break the weak bonds holding RNAP to sigma - RNAP elongates and Sigma stays “Scrunching Model”:
Bacterial Transcription: Elongation
Lose sigma factor:
- RNAP proceeds into elongation without sigma factor
Coding strand: Doesnt interact with DNA at all what we see when we look in genome
Pairing of RNA and DNA:
- 8-9 ntof DNA paired with RNA in active site
Proofreading Capabilities:
Pyrophosphorolytic Editing: amino acids in the RNAP active site can remove the last nt added, functionally equivalent to DNA exo domain Hydrolytic Editing: RNAP can back up >1 base and can remove the RNA to new starting point
Bacterial Transcription: Termination
3 Types of Termination:
Rho-dependent Termination:
requires Rho protein, a ring like hexameric complex with ATPase activity
- Surrounds the nascent RNA
Rho-independent Termination:
In many cases e.coli contains DNA terminator sequences
- As inverted repeat followed by alot of A’s
- As it gets transcribed hairpin forms and forces RNAP off the DNA
- A-U pairs easier to break
TRCF-mediated Diassociation:
- When memorizing termination processes this is one!!
- When RNAP stalls the TRCF promote dissociation
- RNA lost and degraded
Transcription in Eukaryotes
Basics:
-Super claw
- more proteins, regulation, and options
RNAP 1:
Transcribes all large ribosomal RNA
RNAP 2:
mRNA tscript
RNAP 3:
tRNA and 5srRNA subunit
RNA Polymerase II Structure:
“Basically” same as e.coli RNAP
Core Subunits:
Related to beta prime, alpha, beta, and omega
Common Subunits:
Subunits found in all 3 RNAP
Non-essential Subunits:
“congenitally dispensable”
best possible growth condition
Eukaryotic Transcription: Initiation Promoters
Class II Promoters:
Evolved from RNAP 2
1) Initiator seq contains the +1
2) TB2B recognition element
3) TATA box
4) Down stream elements
- +28 reigon and so onto the right
-not much or less is known
- Identifed in mutant screens for mutations that eliminate tscript
Eukaryotic Transcription: Initiation Promoters
Initiator Sequence (Inr):
Some genes have this region at the site of tscript initiation
Consensus: Py-Py-A-N-(T/A)-Py-Py
The TATA Box:
Seq at -25 to -31 nt region
Consensus: 5’ TATAAAA 3’
Downstream Elements:
Little or no consensus known
Eukaryotic Transcription: Initation The Proteins (Transcription Factors)
Pre-initiation Complex:
Complete set of general tscript factors and RNAP bound to the DNA
TFIID:
-General tscript factor protein complex
- binds to TATA box bc it contains a TATA box binding protein (TBP)
TBP-DNA complex:
TBP binds the DNA to facillitate other TF binding
-TBP also binds to other proteins
- 8-10 known TBP-Associated factors (TAF)
TFIIH:
- Uses ATP to unwind/melt double helix
- RNAP 2 cannot do this
Mediator Complex:
- HUGE protein complex w/ >20 subunits amd chromatin
- mechanisms that allow proteins bound far away from the gene to affect tscript
TBP-DNA complex:
Eukaryotic Transcription: Initation Promoter Escape
RNAP II Carboxy Terminal Tail (CTD):
- Starts out as unphosphorylated
- In these states binds to promoter and tscript factors
- When it gets phosphorylated we have promoted escape
- Phosphorylation pattern changes to initiate different states of tscript
Eukaryotic Transcription: Elongation
Elongation Factors:
Proteins evolved in promoting elongation
1) maintain processivity for RNA
2) assist in splicing
CTD: Heptapeptide repeats:
7 peptide repeats
Phosphoryation of Serines:
Primary phosphorylation sites are on serines
Phosphorylation Patters:
1) Unphosphorylated= promotor binding
2) Phosphor of serine 5= promoter escape
3) Of serine 2= promotes RNA splicing
Eukaryotic Transcription: Elongation RNA Capping
What is it?
Capped with a modified (methylated) guainine that is bound by a 5’-5’ triphosphate bridge
When does it occur?
Very early in tscript within the first 30 nt sythnesized
Functions:
1) Protects RNA from degradation
2) Enhances translation (helps ribosomes bind)
3) Enhances transport of mRNA out of nucleus
4) Enhances splicing efficiently at 5’ end
Eukaryotic Transcription: Termination Polyadenylation
What is it?
- Multiple ademines added to the 3’ end of the RNA
- Added without template
When does it occur?
-Close to the end of tscript (end is when RNAP leaves)
Proteins involved:
1.CTP domain bound by proteins called “Poly A” factors
2. CsTF binds poly a signal seq and cleaves nascent RNA
3. CpSF binds signal seq and recruits poly a polymerase (PAP)
4. PAP synthesizes the A tail
Functions:
1) Protects coding seq from being quickly degraded
2) Improve translation of 3’ end mRNA
Eukaryotic Transcription: Termination
When does termination take place?
- Very soon after Poly A tail added
What facilitates termination?
- Torpedo Model: After capped mRNA is cut off, nuclease starts degrading the 5’ end of the remaining RNA –> when it hits RNAP, helps it release from DNA
Intron Splicing via Spliceosome
DNA sequence-mediated:
While splice sites are dependent on DNA sequence, there is actually very little seq requirement- means correct splicing req LOTS of control by other molecules
5’ splice site:
GU at 5’ end
3’ splice site:
AG at 3’ end
Branch site:
Adenine
Chemistry of Splicing
1) 2’ OH of Adenosine in Branch site:
1. Nucelophilic attack of 2’ OH on branch point adenine and last nucleotide on upstream exon
2. Forms larant loop adenine has both 5’-2’ and 5’-3’ phosphidiester bonds
3. 3’ OH of last nucleotide in upstream bond between last nucleotide in intron and 1st nucleotide in downstream exon
3’ OH of 1st exon:
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Spliceosomes
General Structure:
contains more than 150 proteins
small nuclear RNAs:
around 100-300 nt long
in complexes with proteins
small nuclear ribonuclear proteins:
name for individual RNA protein complexes
3 roles:
1) recognize the 5’ splice site
2) bring sites together for rxn
3) catalyze/help catalyze RNA rxn
snRNPs
U1:
Binds to 5’ splice site by complementary base pairing
U2:
Base pairs w/sequences at intron branch site and then associates with U6
U6:
Binds to 5’ splice site by base pairing after U1 and associates U2
U5:
Associates with the last nucleotide of upstream exon and 1st nucleotide of downstream intron and positions them together
U4:
Associates with u6 until u6 is needed and then disassociates
The Splicing Pathway
Starting Point:
RNA before spliced pre-mRNA
Step 1:
A) U1 binds 5’ splice site
B) BBP binds the branch site which contains branch-point adenine
C)——-
Step 2:
U2 displaces BBP at branch site
Step 3:
U4,U5 and U6 bind each other
Tri-snRNP complex -floats around until it binds to U1 and U2 at introns (weak bonds)
Step 4:
Tri-snRNP complex binds U1 and 2
The Splicing Pathway (continuted)
Step 5:
Step 6:
Step 7:
Step 8
Self-splicing RNAs (are they ribozymes?)
Self-splicing:
Group II introns:
Group I introns:
Splicing Mechanism:
1)
2)
3)
Splicing Errors (and how they are avoided)
Possible Errors:
1) Exon skipping 2) Pseudo splice-site selection
Splicing Fidelity Mechanisms:
1) Co-transcriptional loading 2) Splicing “guides”
Alternative Splicing
Definition:
Regulated process:
Human Slo gene:
Drosophila Dscam gene
Interesting Splicing Phenomena
Trans-Splicing:
RNA Editing:
Cytidine deaminase and apolipoprotein B:
More Interesting RNA Modifications
RNA Editing:
Cytidine deaminase and apolipoprotein B
General Points
Formal definition:
Energetic cost:
4 Primary Components: 1)
2)
3)
4)
The messenger RNA
Terminology Review
ORF:
monocistronic:
Polycistronic:
Sites of Translation Initiation
In Prokaryotes:
In Eukaryotes:
transfer RNAs (tRNAs)
Role:
Common Structural Elements: 1)
2)
3)
Examples of unusual nucleotides:
Aminoacyl-tRNA synthetase
Function:
Steps in Charging of tRNA
1)
2)
Accuracy:
Ribosome
Composition:
Subunits
Large: Small:
Terminology:
Svedberg Units:
Prokaryotes Eukaryotes Large: Small: Combined:
Ribosome: Sites
3 Sites to know
A site:
P site:
E site:
Stages of Translation
Initiation:
Elongation:
Termination
Translation Initiation: Prokaryotes
Important Components:
1) 2) 3) 4)
Translation Initiation: Prokaryotes
The Process
Initial State:
Step 1:
IF3:
Step 2:
IF1:
IF2:
Translation Initiation: Prokaryotes
The Process (continued)
Step 3)
The Process (continued)
Step 4)
Step 5)
Translation Termination
Stop codon recognition:
Release Factors:
Class I: Class II:
End result for ribosome
