Translation

1
Translation
2
Translation

• Translation- the synthesis of protein from an RNA
  template.

Five stages Preinitiation Initiation Elongatio
n Termination Post-translational modification
3
Translation Is the Most Complicated Biological
Process Known

• In eukaryotes,
• gt70 ribosomal proteins
• 20 (more) proteins to activate aas
• 12 (more) auxiliary enzymes
• 100 proteins for processing
• 40 tRNA and rRNAs (minimum)
• Other specific proteins
• 300 molecules involved

4
Translation

• Importance
• Location ribosomes (predominantly Cp)
• Highly regulated
• Fast 20 aa/sec

5
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

6
The Genetic Code has been Cracked

• Poly U codes for poly Phe

7
The Genetic Code has been CrackedTriplet Codons
Code for a Single Amino Acid

• UUU Phe

8
The Genetic Code has been CrackedThe Code is
Commaless

• AUGUUU
• Met Phe

9
The Genetic Code has been CrackedThe Code is
Nonoverlapping

• AUGUUU AUGUUU
• Met Phe Not Met Cys Val

10
The Genetic Code has been CrackedThe Code is
Redundant

• 1 codon for Met, Trp
• all other amino acids have 2-6 triplets

11
The Genetic Code has been Cracked The Code is
Degenerate

• UCU Ser
• UCC Ser
• UCA Ser
• UCG Ser
• Wobble exists in the third position

12
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 oneCG 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

13
The Genetic Code has been Cracked The Code is
Unambiguous

• UUU doesnt code for Phe sometimes, and other
  times Ser

14
The Genetic Code has been Cracked The Code is
Universal

• Not completely true
• Mitochondria, e.g., have some codons different
• Nuance is codon usage

15
The Genetic Code has been Cracked Three Reading
Frames are Possible

• ACUGUCGCUC...
• One ACU GUC GCU C...
• Two CUG UCG CUC...
• Three UGU CGC UC...
• (Four) GUC GCU C...

16
The Genetic Code has been Cracked Embedded Genes
are PossibleFig. 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.

17
The Genetic Code has been Cracked Nonsense
Codons are Stop Points

• UAA, UAG, and UGA

18
mRNA Structure

• There is a 5UTR
• Shine-Dalgarno sequence (prokaryotes)
• 10 nt upstream of initiation codon
• Consensus 4-9 Pu 8-13 nt upstream
• Positions ribosome at correct start site

19
All tRNAs Have a Similar but not Identical
Structure Cloverleaf

• 75 nt
• Acceptor arm- CCA-3
• T?C arm
• Variable length extra arm 3-20 nt
• anti-codon arm
• D arm
• canonical positions
• Identity elements

20
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

21
Components of Mammalian Ribosomes(After Harpers
Biochemistry)
22
rRNA StructureScientific American circa 1960 and
Science 2002
23
The P Site and the A SiteFig. 27-11 Lehninger
POB 3rd Ed.
24
The Preinitiation Stage is Characterized by
Formation of Required Starting Complexes
25
Preinitiation - Charging the tRNA
26
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

27
Mechanism of Class I vs. Class IIFig. 27-14
Lehninger POB 4th Ed.
28
Perspective on ScaleFig. 27-17(a) Lehninger POB
4th Ed.
29
Preinitiation Formylation of met-tRNAfmet
(Prokaryotes Only)
30
Preinitiation Dissociation of Ribosomes (IF-1
and IF-3)
31
Preinitiation IF-2GTP Binary Complex Formation

• IF-2B represents a guanine exchange molecule

32
Preinitiation IF-2GTPCharged tRNA Ternary
Complex Formation
33
Preinitiation 40S Preinitiation Complex
34
Initiation IF-4F, 4A and 4B Bind mRNA to Place
it on Small Subunit
35
Initiation 40S Initiation Complex
36
Initiation 80S Initiation Complex
37
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

38
Elongation

• 1. EF-1GTPaminoacyl-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

39
Elongation I
40
Elongation Peptide Bond Forms as P-site Content
is Transferred Onto A-Site Occupant
41
Mechanism of Peptide Bond Formation
42
Mechanism of Peptide Bond Formation
43
Elongation Translocation Requires GTP-bound EF-2
44
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

45
Termination UAA, UAG, or UGA is Enveloped by
A-site of Ribosome
46
Termination RF-1 Enters A Site
47
Termination GTP is Hydrolyzed and H2O is Used
to Cleave Protein Off tRNA
48
Energetics

• Each amino acid residue requires gt4 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

49
Regulation of Translation eEF 2

• a. eEF 2 phosphorylated under stress
• b. when phosphorylated, eEF 2B doesnt allow
  GDP-GTP exchange and protein synthesis stops

50
Regulation of Translation eIF 4F

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

51
Post-translational Modifications

• 1. Proteolytic cleavage (most common)
• Signal sequences zymogens
• 2. Disulfide bond formation not as well
  understood
• 3. Group addition
• a. Glycosylation (most complex known)
• b. Acetylation or phosphorylation, etc.
• c. Farnesyl or Geranylgeranyl
• 4. Amino acid modification
• a. Hydroxylation of Pro (in ER)
• b. Methylation of Lys
• 5. Other things

52
Signal SequenceHuman 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

53
SRP Ribosome CycleFig. 27-33 Lehninger POB 4th
Ed.
54
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

55
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

56
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
• Doesnt have to be protein signal M6P targets
  to lysosome

57
Other Proteolytic Events

• Met aminopeptidase
• Proprotein vs preproprotein

58
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/us and somehow facilitate appropriate
  interactions

59
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

60
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

61
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)

62
Protein Degradation The Ultimate in
Posttranslational Modification

• Regulated
• 1. The N-end Rule
• not a sensitive system

63
The N-End Rule
64
Protein Degradation

• Regulated
• 1. The N-end Rule
• not a sensitive system
• 2. Lysosome
• endocytosis

3. The Ubiquitin system
65
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

66
More Than You Ever Wanted to Know About
UbiutinScience 315 201-205, 2007

• Monoubiquitination- a single Ub is attached
• Multiubiquitination- several residues of target
  protein each have single Ub is attached
• Polyubiquitination- numerous copies of Ub
  attached to same site of target protein. Ub has
  7 different Lys, so many ways to do this. Once
  polyUbd, targeted for destruction
• Ub can be removed from protein
• So clearly there must be mechanism to determine
  level of Ub

67
Ub is Now Known to Do More Than Just Target for
DestructionScience 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.

68
Ub is Now Known to Do More Than Just Target for
DestructionScience 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
  proteincoupled 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.

69
Ub is Now Known to Do More Than Just Target for
DestructionScience 315 201-205, 2007