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: