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