Exam 3

Question 1

RNA Splicing: An Introduction

Answer 1

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.

Question 2

Interesting Facts about Introns

Answer 2

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

Question 3

Evolutionary Implications of RNA Splicing

Answer 3

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

Question 4

Intron Splicing Classes

Answer 4

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

Question 5

What is transcription?

Answer 5

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)

Question 6

RNA Polymerase: The Claw

Answer 6

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

Question 7

Main Steps of Bacterial Transcription

Answer 7

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)

Question 8

Introducing DNA Consensus Sequences: When You’re Trying To Find a Pattern That Might Mean Something Biological

Answer 8

Sequence Alignment:
Consensus Sequence:

Question 9

Bacterial Transcription: Initiation

Answer 9

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

Question 10

Sigma Factors (σ70, σ32, σ54…)

Answer 10

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

Question 11

Bacterial Transcription: Initiation (continued)

Answer 11

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”:

Question 12

Bacterial Transcription: Elongation

Answer 12

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

Question 13

Bacterial Transcription: Termination

Answer 13

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

Question 14

Transcription in Eukaryotes

Answer 14

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

Question 15

Eukaryotic Transcription: Initiation Promoters

Answer 15

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

Question 16

Eukaryotic Transcription: Initiation Promoters

Answer 16

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

Question 17

Eukaryotic Transcription: Initation The Proteins (Transcription Factors)

Answer 17

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

Question 18

TBP-DNA complex:

Question 19

Eukaryotic Transcription: Initation Promoter Escape

Answer 19

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

Question 20

Eukaryotic Transcription: Elongation

Answer 20

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

Question 21

Eukaryotic Transcription: Elongation RNA Capping

Answer 21

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

Question 22

Eukaryotic Transcription: Termination Polyadenylation

Answer 22

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

Question 23

Eukaryotic Transcription: Termination

Answer 23

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

Question 24

Intron Splicing via Spliceosome

Answer 24

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

Question 25

Chemistry of Splicing

Answer 25

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:

>

*)

**)

Question 26

Spliceosomes

Answer 26

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

Question 27

snRNPs

Answer 27

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

Question 28

The Splicing Pathway

Answer 28

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

Question 29

The Splicing Pathway (continuted)

Answer 29

Step 5:

Step 6:

Step 7:

Step 8

Question 30

Self-splicing RNAs (are they ribozymes?)

Answer 30

Self-splicing:

Group II introns:

Group I introns:

Splicing Mechanism:
1)

2)

3)

Question 31

Splicing Errors (and how they are avoided)

Answer 31

Possible Errors:

  1)  Exon skipping

2) Pseudo splice-site selection

Splicing Fidelity Mechanisms:

1)  Co-transcriptional loading

2)  Splicing “guides”

Question 32

Alternative Splicing

Answer 32

Definition:

Regulated process:

Human Slo gene:
Drosophila Dscam gene

Question 33

Interesting Splicing Phenomena

Answer 33

Trans-Splicing:

RNA Editing:

Cytidine deaminase
and apolipoprotein B:

Question 34

More Interesting RNA Modifications

Answer 34

RNA Editing:

Cytidine deaminase
and apolipoprotein B

Question 35

General Points

Answer 35

Formal definition:

Energetic cost:

4 Primary Components: 1)
2)
3)
4)

Question 36

The messenger RNA

Answer 36

Terminology Review

ORF:

monocistronic:

Polycistronic:

Sites of Translation Initiation

In Prokaryotes:

In Eukaryotes:

Question 37

transfer RNAs (tRNAs)

Answer 37

Role:

Common Structural Elements: 1)
2)
3)
Examples of unusual nucleotides:

Question 38

Aminoacyl-tRNA synthetase

Answer 38

Function:

Steps in Charging of tRNA

1)

2)

Accuracy:

Question 39

Ribosome

Answer 39

Composition:

Subunits

 Large:

 Small:

Terminology:
Svedberg Units:

                 Prokaryotes    Eukaryotes
  Large:	
  Small:
  Combined:

Question 40

Ribosome: Sites

Answer 40

3 Sites to know

A site:

P site:

E site:

Question 41

Stages of Translation

Answer 41

Initiation:

Elongation:

Termination

Question 42

Translation Initiation: Prokaryotes

Answer 42

Important Components:

1)  

2)

3)

4)

Question 43

Translation Initiation: Prokaryotes

Answer 43

The Process

Initial State:

Step 1:
IF3:

Step 2:
IF1:
IF2:

Question 44

Translation Initiation: Prokaryotes

Answer 44

The Process (continued)

Step 3)

The Process (continued)

Step 4)

Step 5)

Question 45

Translation Termination

Answer 45

Stop codon recognition:

Release Factors:

Class I:

Class II:

End result for ribosome