RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
methylated guanine “backward” 5′ to 5′ linkage Not encoded in DNA Capping enzyme Recognition by ribosome
5′ cap
5′ AGACCUGACCAUACC
RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template Important for:
Export of mRNA Initiation of Translation Stability of mRNA
…UGGCAGACCUGACCA 3′
…UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA
RNA processing in eukaryotes
DNA
promoter
exons introns primary transcript(nucleus)
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
transcriptiontranscription
unbroken coding sequence
transport to cytoplasm for translationtransport to cytoplasm for translation
final mRNA
Splicing Most genes interrupted by introns Introns removed after transcription Exons spliced together
5’ capAAAAAAAAA3’ poly-A tail
AAAAAAAAA
splicingsplicing
unbroken coding sequencefinal mRNA
Splicing snRNPs recognize exon-intron
boundaries RNA + protein Cut and rejoin mRNA
Splicing
RPE65 mRNA in nucleus: 21,000 nt (14 exons)
AAAAAAAAA
AAAAAAAAA
splicingsplicing
mature RPE65 mRNA in nucleus: 1,700 nt (8%)
Splicing Alternative splicing: >1 protein from one gene 27,000 human genes, but >100,000 proteins
Splicing
Mutations affecting splicing can cause genetic disease:cystic fibrosis retinitis pigmentosaspinal muscular atrophy Prader-Willi syndromeHuntington disease spinocerebellar ataxiamyotonic dystrophy Fragile-X syndrome
Or produce genetic susceptibility to disease:lupus bipolar disorderschizophrenia myocardial infarctiontype I diabetes asthmacardiac hypertrophy multiple sclerosisautoimmune diseases elevated cholesterol
Gene expression summary
Prokaryotes Eukaryotes
DNA
mRNA
directly translated(even before beingcompletely transcribed)
transcription
protein
cyto
pla
sm
DNA
pre-mRNA• capping• polyadenylation• splicing
transcription
mature mRNA
protein
• transport to cytoplasm• translation
cyto
pla
smnucl
eus
Quick review of protein structure amino acids
C CNH2
R
H
OH
O
generic amino acid
Quick review of protein structure side chain gives chemical properties
C CNH2
R
H
OH
O
Charged:Negative:
Polar, not charged:
Non-polar (hydrophobic):
Positive:
Quick review of protein structure polymer of amino acids = polypeptide ≈ protein
methionine aspartate
C CNH2
CH2
H
OH
O
CH2
S
CH3
C CNH2
CH2
H
OH
O
C
O
OH
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
OH
OH
C
O
OH
Quick review of protein structure polymer of amino acids = polypeptide ≈ protein
methionine aspartate
peptide bond
N-terminus
C-terminus
polymer of amino acids = polypeptide ≈ protein
Quick review of protein structure
methionine aspartate glycine
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
N
OH
C
O
OH
C C
H
H
OH
OH
polymer of amino acids = polypeptide ≈ protein
Quick review of protein structure
methionine aspartate glycine phenylalanine
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
N
OH
C
O
OH
C C
H
H
N
OH
C C
CH2
H
OH
OH
polymer of amino acids = polypeptide ≈ protein
Quick review of protein structure
methionine aspartate glycine phenylalanine
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
N
OH
C
O
OH
C C
H
H
N
OH
C C
CH2
H
N
OH
C C
CH
H
OH
OH
CH3 CH3
valine
polymer of amino acids = polypeptide ≈ protein
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
N
OH
C
O
OH
C C
H
H
N
OH
C C
CH2
H
N
OH
C C
CH
H
N
OH
CH3 CH3
C C
CH2
H
OH
OH
CH2
CH2
CH2
NH2
Quick review of protein structure
methionine aspartate glycine phenylalanine valine lysine
What holds folded proteins together?Hydrogen bondsHydrophobic interactions Ionic bondsDisulfide bonds (covalent)
…all determined by amino-acid sequence
Quick review of protein structure
Primary (1°) structure
Quick review of protein structure
C CNH3+
CH2
H
N
O
CH2
S
CH3
C C
CH2
H
N
OH
C
O
OH
C C
H
H
N
OH
C C
CH2
H
N
OH
C C
CH
H
N
OH
CH3 CH3
C C
CH2
H
OH
OH
CH2
CH2
CH2
NH2
Secondary (2°) structure
Tertiary (3°) structure
Quaternary (4°) structure
betasheet
alphahelix
L-isoaspartylprotein carboxyl methyltransferase
hemoglobin
Translation Ribosome finds start codon within mRNA Genetic code determines amino acids Stop codon terminates translation
translation
NH3 COOHprotein
5′ UTR
coding region
startcodon
stopcodon
3′ UTR
5′ 3′mRNA
Ribosome Large ribonucleoprotein structure
E. coli: 3 rRNAs, 52 proteins Two subunits: large and small
RNA
largesubunit
smallsubunit
protein
Eukaryotic Translation How does the ribosome find the correct start codon?
Small ribosome subunit binds 5 cap′ Scans to first AUG
5′ UTR
coding region
startcodon
stopcodon
3′ UTR
5′ 3′mRNAcap
AAAAAAAAA…
Prokaryotic Translation How does the ribosome find the correct start codon?
Small subunit binds Shine-Dalgarno sequence (RBS) Positioned correctly for translation
5′ UTR
coding region
startcodon
stopcodon
3′ UTR
5′ 3′mRNA
Shine-Dalgarno sequenceor RBS (AGGAGG)
After finding start codon, use the genetic code:
Shown as mRNA 5 ′ → 3′
the Genetic Code
Mechanics of Translation Translation requires:
mature mRNA ribosome tRNAs amino acids accessory proteins
tRNA
anticodon
Small RNAs (74-95 nt) made by transcription Intramolecular base pairing Anticodon complementary to mRNA codon
tRNA “Charged” by specific aminoacyl tRNA synthetase
Initiation of Translation Small ribosome subunit binds at start codon
Prokaryotes: Shine-Dalgarno sequence (RBS) Eukaryotes: binds cap, scans
mRNA5′ AUG GAU GGG
Initiation of Translation First tRNA (Met, anticodon CAU) joins complex
AUG
5'3'
Met
UACGAU GGG
mRNA5′
Initiation of Translation Large ribosomal subunit joins
AUG
5'3'
Met
UACGAU GGG
mRNA5′
Initiation of Translation P site holds tRNA with first aa A site open for next tRNA
P A
AUG
5'3'
Met
UACGAU GGG
mRNA5′
Initiation of Translation
Elongation Next tRNA enters
P A
AUG
5'3'
Met
UACGAU
5'3'
Asp
CUA
GGGmRNA
5′
Elongation Peptidyl transferase forms peptide bond
Amino acid released from tRNA in P site
AUG
5'3'
Met
UACGAU
5'3'
Asp
CUAGGG
Met
mRNA5′
Elongation Ribosome translocates one codon
First tRNA binds briefly in E site until translocation completes
AUG
5'3'UAC
GAU5'3'
Asp
CUAGGG
Met
mRNA5′
Elongation Process repeats
Next tRNA can then enter the empty A site
AUG GAU5'3'
Asp
CUAGGG
Met
mRNA5′
P A
5'3'
Gly
CCC
Elongation
Termination Ribosome stops at stop codon
No matching tRNA Release factor binds
UUG CAG5'3'
Gln
GUCUAG
Leu P AAspMet Gly Phe Val Lys Gly Asp Ile Leu Val
RF
Translation complex dissociates
Termination
UUG CAG
Gln
5'3'GUC
UAG
LeuAspMet Gly Phe Val Lys Gly Asp Ile Leu Val
RF
Polyribosomes Next ribosome starts as soon as start codon is available
Releasedpolypeptide
Growingpolypeptide
5' – 3' direction ofribosome movement
Stop
RNA subunitsreleased
Ribosome
mRNAAUG
5'3'
N
CN
Operons More than one gene on one mRNA Prokaryotes only
Operons More than one gene on one mRNA Prokaryotes only
Protein Synthesis Pathways Free ribosomes Ribosomes bound to RER