Translation

Translation

Translation- the synthesis of protein from an RNA template.

Five stages:

Preinitiation

Initiation

Elongation

Termination

Post-translational modification

Translation Is the Most Complicated Biological Process Known

In eukaryotes,>70 ribosomal proteins20 (more) proteins to activate aa’s12 (more) auxiliary enzymes≥100 proteins for processing≈40 tRNA and rRNAs (minimum)Other specific proteins ~300 molecules involved

Translation

• Importance

• Location: ribosomes (predominantly Cp)

• Highly regulated

• Fast: ~20 aa/sec

Functions of the Types of RNA

mRNA- serves as a template code

tRNA- serves as an adapter molecule

rRNA- holds molecules in the correct position, protein portion also catalyze reactions

The Genetic Code has been Cracked

Poly U codes for poly Phe

The Genetic Code has been CrackedTriplet Codons Code for a Single Amino

AcidUUU = Phe

The Genetic Code has been CrackedThe Code is Commaless

AUGUUU

Met Phe

The Genetic Code has been CrackedThe Code is Nonoverlapping

AUGUUU AUGUUU

Met Phe Not Met Cys Val

The Genetic Code has been CrackedThe Code is Redundant

1 codon for Met, Trp

all other amino acids have 2-6 triplets

The Genetic Code has been Cracked The Code is Degenerate

UCU Ser

UCC Ser

UCA Ser

UCG Ser

“Wobble” exists in the third position

The Wobble Hypothesis

• First two bases of codon form strong h-bonds• If 1st base of anticodon is C or A (i.e. last base

of codon is G or U), only one:CG and AU base pairs formed

• If 1st base of anticodon is G or U two codons can be recognized: U to A or G; G to C or U

• If 1st base of anticodon is I, three codons recognized: I to A, U or C

• By not having to verify all three bases, speed up process

The Genetic Code has been Cracked The Code is Unambiguous

UUU doesn’t code for Phe sometimes, and other times Ser

The Genetic Code has been Cracked The Code is Universal

Not completely true

Mitochondria, e.g., have some codons different

Nuance is codon usage

The Genetic Code has been Cracked Three Reading Frames are Possible

ACUGUCGCUC...

One: ACU GUC GCUC...

Two: CUG UCG CUC...

Three: UGU CGC UC...

(Four):GUC GCU C...

The Genetic Code has been Cracked Embedded Genes are Possible

Fig. 26-3 Lehninger POB 4th Ed.

Open Reading Frame- ORF a long enough sequence between start and stop to code for legitimate protein.

In viral systems, there are also examples of overlapping genes.

The Genetic Code has been Cracked Nonsense Codons are Stop Points

UAA, UAG, and UGA

There is a 5’UTR

Shine-Dalgarno sequence (prokaryotes)

~10 nt upstream of initiation codon

Consensus: 4-9 Pu 8-13 nt upstream

Positions ribosome at correct start site

mRNA Structure

All tRNA’s Have a Similar but not Identical Structure: “Cloverleaf”

• ~75 nt• Acceptor arm- CCA-3’• TC arm• Variable length extra

arm ~3-~20 nt• anti-codon arm• D arm• canonical positions• Identity elements

rRNA Structure (E. coli Because Well-Known)

• Small subunit (30S)– 16S RNA– 21 proteins (S1-S21)

• Large subunit (50S)– 5S and 23S RNA– 36 proteins (L1-L36)

• Combined = 70S

• Eukaryote 40S + 60S = 80S

Components of Mammalian Ribosomes(After Harper’s Biochemistry)

Component Mass Protein RNA

No. Mass Size Mass Bases

40S s/u 1.4x106 ~35 7x105 18S 7x105 1900

60S s/u 2.8x106 ~35 7x105 18S 7x105 1900

~50 1x106 5S 35,000 120

5.8S 45,000 160

28S 1.6x106 4700

rRNA StructureScientific American circa 1960 and Science 2002

The P Site and the A SiteFig. 27-11 Lehninger POB 3rd Ed.

The Preinitiation Stage is Characterized by Formation of Required Starting Complexes

Preinitiation - Charging the tRNA

OCH2

OH OH

NN

N N

NH2

O

P

O

OCCtRNA

C H

R

H3N

C O

OCH2

O OH

NN

N N

NH2

O

P

O

OCCtRNA

ATP PPi

CO2

C H

R

H3N

AMP

Aminoacyl-tRNAsynthetase

.

.

Aminoacyl-tRNA Synthetase

• One for each amino acid• 2 step mechanism:

1. attach a.a. to AMP

2a. transesterify to 3’ (class 2)

2b. transesterify to 2’ and then rearrange(class 1)

• Proofread– identity elements– “sieve”

Mechanism of Class I vs. Class IIFig. 27-14 Lehninger POB 4th Ed.

Perspective on ScaleFig. 27-17(a) Lehninger POB 4th Ed.

Preinitiation: Formylation of met-tRNAfmet (Prokaryotes Only)

met-tRNAfmetfmet-tRNAfmetFormyltransferase

formyl-THF THF

.

.

Preinitiation: Dissociation of Ribosomes (IF-1 and IF-3)

Preinitiation: IF-2:GTP Binary Complex Formation

• IF-2B represents a guanine exchange molecule

Preinitiation: IF-2:GTP:Charged tRNA Ternary Complex Formation

Preinitiation: 40S Preinitiation Complex

Initiation: IF-4F, 4A and 4B Bind mRNA to Place it on Small Subunit

Initiation: 40S Initiation Complex

Initiation: 80S Initiation Complex

Initiation

Preinitiation complexes form an 80S complex:

small subunit, ternary complex (GDP + Pi leave), mRNA, large subunit, aminoacyl tRNA

P-site- only thing that can enter is a peptide

In prokaryotes, f-met “tricks” the ribosome

A-site- only thing that can enter is an aminoacyl tRNA

Elongation

1. EF-1:GTP:aminoacyl-tRNA ternary complex enters A-site; GDP + Pi leave

(EF-Tu and EF-Ts involved with GTP metabolism in prokaryotes)

2. Peptide bond forms as P-site content is transferred onto A-site occupant

3. Translocation requires GTP-bound EF-2

Elongation I

Elongation: Peptide Bond Forms as P-site Content is Transferred Onto A-Site

Occupant

Mechanism of Peptide Bond Formation

OO C

O

C

NH2

H

R1

OHA

tRNA CC

OO C

O

C

NH2

H

R2

OHA

tRNA CC

P Site

A Site

.

.

Mechanism of Peptide Bond Formation

OOH

OHA

tRNA CCP Site

A Site

C C

NH3

H

R1O

OO C

O

C

N

H

R2

OHA

tRNA CCH

.

.

Elongation: Translocation Requires GTP-bound EF-2

Termination

1. UAA, UAG, UGA is enveloped by A-site of ribosome

2. RF-1 enters A site

3. GTP is hydrolyzed, H2O is used to cleave protein off tRNA

4. Components are recycled to synthesize another protein molecule

Termination: UAA, UAG, or UGA is Enveloped by A-site of Ribosome

Termination: RF-1 Enters A Site

Termination: GTP is Hydrolyzed and H2O is Used to Cleave Protein Off tRNA

Energetics

Each amino acid residue requires >4 ATP equivalents

ATP→AMP + PPi to “charge” tRNA

1 GTP is used to place aminoacyl-tRNA into A-site

1 GTP is used to translocate after each peptide bond formation

ATP hydrolysis for unknown purpose at…

Regulation of Translation: eEF 2

a. eEF 2 phosphorylated under stress

b. when phosphorylated, eEF 2B doesn’t allow GDP-GTP exchange and protein synthesis stops

Regulation of Translation: eIF 4F

4F is complex of 4E (cap binding protein) and 4A (ATPase that unwinds RNA) and 4G (function unknown)

a. eIF-4E can be phosphorylated: why or how unknown- but this activates

b. 4E-BP complex forms which inactivates 4E. Phosphorylation in presence of insulin dissociates complex

Post-translational Modifications

1. Proteolytic cleavage (most common)Signal sequences; zymogens

2. Disulfide bond formation: not as well understood3. Group addition

a. Glycosylation (most complex known)b. Acetylation or phosphorylation, etc.c. Farnesyl or Geranylgeranyl

4. Amino acid modificationa. Hydroxylation of Pro (in ER)b. Methylation of Lys

5. Other things

Signal Sequence:Human PreProInsulin

Met Ala Leu Trp Met Arg(+) Leu Leu Pro

Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro

Asp Pro Ala Ala Ala Phe Val

SRP Ribosome CycleFig. 27-33 Lehninger POB 4th Ed.

Other Signal SequencesFig. 27-39 Lehninger POB 3rd Ed.

• KDEL anywhere in protein directs back to ER

• Chloroplast and Mitochondria:– Different in that whole

protein is made and then directed via chaperone proteins

– Different sequences: mitochondrial not contiguous

Other Signal SequencesFig. 27-37 Lehninger POB 4th Ed.

• KDEL anywhere in protein directs back to ER

• Chloroplast and Mitochondria:– Different in that whole protein

is made and then directed via chaperone proteins

– Different sequences: mitochondrial not contiguous

• Nuclear signal:– Different in that not cleaved

Other Signal Sequences

• KDEL anywhere in protein directs back to ER

• Chloroplast and Mitochondria:– Different in that whole protein

is made and then directed via chaperone proteins

– Different sequences: mitochondrial not contiguous

• Nuclear signal:– Different in that not cleaved

• Doesn’t have to be protein signal: M6P targets to lysosome

Other Proteolytic Events

• Met aminopeptidase

• Proprotein vs preproprotein

Disulfide Bond Formation is Not Well Understood

• Occurs in ER

• PDI and Chaperones involved

• PDI “shuffles” disulfide bonds

• Chaperones consist of two rings of identical s/u’s and somehow facilitate appropriate interactions

Glycosylation Occurs in Multiple Compartments (ARB figure)

Oligo “tree” transferred en bloc from dolichol-based structure

Some trimming takes place before export from ER

Often, further trimming in cis-Golgi…

Glycosylation Continued

…before building up with different sugars takes place

Packaging occurs as the proteins pass through the trans-golgi network

Exocytosis into bloodstream takes place

Microheterogeneity

Lipid MoietiesFig. 27-30 Lehninger POB 4th Ed.

• CaaX farnesylation signal (a for aliphatic)• aaX removed after farnesyl added• CXX or CXC geranylgeranylation signal (X for

anything)

Protein Degradation: The Ultimate in Posttranslational Modification

Regulated:

1. The N-end Rule

not a sensitive system

The N-End Rule

Protein Degradation

Regulated:

1. The N-end Rule

not a sensitive system

2. Lysosome

endocytosis3. The Ubiquitin system

UbiquinationFig. 27-41 Lehninger POB 4th Ed.

• Ubiquitin common throughout the animal kingdom

• 76 aa (8.5 kDa) i.e. small• 53% homology between yeast

and humans

• E1 (ubiquitin activating enzyme)• E2 (ubiquitin carrier protein)• E3 (ubiquitin protein ligase) selects proteins to tag by N-term

More Than You Ever Wanted to Know About UbiutinScience 315: 201-205, 2007

Monoubiquitination- a single Ub is attachedMultiubiquitination- several residues of target

protein each have single Ub is attachedPolyubiquitination- numerous copies of Ub attached

to same site of target protein. Ub has 7 different Lys, so many ways to do this. Once polyUb’d, targeted for destruction

Ub can be removed from proteinSo clearly there must be mechanism to determine

level of Ub

Ub is Now Known to Do More Than Just Target for Destruction

Science 315: 201-205, 2007

• In yeast, monoUb is sufficient as an endocytic internalization signal (less clear in animals)– necessary, sufficient

• When epidermal growth factor receptor (EGFR) was stimulated at low epidermal growth factor (EGF) concentrations in HeLa cells, EGFR ubiquitination was not detected, and the receptor localized with clathrin; however, at high EGF concentrations, EGFR was ubiquitinated, and the receptor localized with both caveolae and clathrin.

Ub is Now Known to Do More Than Just Target for Destruction

Science 315: 201-205, 2007

• Endocytosis of certain membrane proteins seems to require polyubiquitination. For example, in the case of the ß2AR, (recall this is a G protein–coupled receptor), agonist stimulation leads to rapid polyubiquitination of both the receptor and the receptor regulatory protein ß-arrestin.

• ...anthrax toxin triggers ubiquitination of its receptor to facilitate efficient and rapid endocytosis of the toxin-receptor complex. ... important for toxin action, because passage through low-pH endosomal compartments makes the toxin competent to induce toxicity in cells

• Protein ubiquitination is best compared to protein phosphorylation.

Ub is Now Known to Do More Than Just Target for Destruction

Science 315: 201-205, 2007