1. Protein Synthesis and Degradation
Chapter 33
Biochemistry 432/832
Protein Synthesis and Degradation
December 10 and 12

2. Announcements

3. Outline 33.1 Ribosome Structure and Assembly
33.2 Mechanics of Protein Synthesis
33.3 Protein Synthesis in Eukaryotes
33.4 Inhibitors of Protein Synthesis
33.5 Protein Folding
33.6 Post-Translational Processing of Proteins
33.7 Protein Degradation

4. Location of tRNA binding sites in a ribosome

5. Peptide chain elongation

6. Reaction of the tRNA-linked peptidyl chain with the a-amino group of an adjacent aminoacyl-tRNA -
no energy is required for activation

7. Peptidyl Transferase This is the central reaction of protein synthesis
23S rRNA is the peptidyl transferase!
The "reaction center" of 23S rRNA - the catalytic bases are among the most highly conserved in all of biology.
Translocation of peptidyl-tRNA from the A site to the P site follows

8. Ribosome is a ribozyme (catalytic rRNA)
Catalytic center is located in the 50S particle
The peptidyl transferase center of 23S rRNA

9. Ribosome is ribozyme
Puglisi JD, Blanchard SC, Green R, Nat Struct Biol 2000 Oct;7(10):855 Approaching translation at atomic resolution.
Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction
In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme
In the 30S structure, tRNA binding sites were located
The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis

10. Ribosome structure
Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF.
Science 2001 Mar 29
Crystal Structure of the Ribosome at 5.5 A Resolution.
We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound mRNA and tRNAs at 5.5 A resolution. All of the 16S, 23S and 5S rRNA chains, the A-, P- and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 A movements associated with tRNA translocation with intersubunit movement.

11. Movement of tRNAs during translation

12. Relative positions of tRNA molecules in a ribosome during peptidyl transfer and translocation

13. Peptide Chain Termination
Proteins known as "release factors" recognize the stop codon at the A site
Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier

14. Termination of protein synthesis

15. The ribosome life cycle

16. Polyribosomes (polysomes) - multiple ribosomes translating the same mRNA molecule

17. The E.coli trp operon Transcription 4 min
Translation of the first two genes 2.5 min

18. Transcription and translation occur at the same time

19. Eukaryotic Protein Synthesis
the structure of the typical mRNA transcript
the 5'-methyl-GTP cap - essential for ribosomal binding, also enhances stability of mRNA
and the poly A tail - enhances stability, enhances translation efficiency
Initiation of protein synthesis in eukaryotes involves a family of at least 11 eukaryotic initiation factors
The initiator tRNA is a special one that carries only Met and functions only in initiation - it is called tRNAiMet but it is not formylated

20. Structure of eukaryotic mRNA
Cap
5’-UTR
Coding region
3’-UTR
Poly-A
Termination (AUG, UGA, UAA)
Initiation (AUG)

21. Eukaryotic Initiation
No Shine-Dalgarno sequences
Begins with formation of ternary complex of eIF-2, GTP and Met-tRNAiMet
This binds to 40S ribosomal subunit:eIF-3:eIF4C complex to form the 43S preinitiation complex
No mRNA yet, so no codon association with Met-tRNAiMet
mRNA then adds with several other factors, forming the initiation complex
ATP is required!

22. Eukaryotic Initiation
Proteins of the initiation complex apparently scan to find the first AUG (start) codon
Not all AUG codons can serve as initiators
Kozak consensus sequence: Pu-Py-Py-A-U-G-Pu (the best at ACCAUGG)

23. Initiation of translation in eukaryotes

24. Initiation of translation in eukaryotes - role of factor eIF4G as a multipurpose adapter to organize 40S, cap, polyA and other factors

25. Regulation of Initiation
Phosphorylation is the key, as usual
At least two proteins involved in initiation (Ribosomal protein S6 and eIF-4F) are activated by phosphorylation
But phosphorylation of eIF-2a causes it to bind all available eIF-2B and sequesters it

26. eIF2 is regulated through reversible phosphorylation of a Ser residue

27. Elongation of peptide chain in eukaryotes
Very similar to prokaryotes
Elongation factor EF1A is equivalent to EF-Tu
binds aminoacyl-tRNA and GTP
Elongation factor EF1B is equivalent to EF-Ts
exchanges GDP with GTP in EF1A
Elongation factor EF2 is equivalent to EF-G
involved in ribosome translocation (GTP-dependent)

28. Termination of protein synthesis in eukaryotes
Only one release factor (RF), homodimer of 55 kDa subunits (three RFs are in prokaryotes)
Binds at the A site
GTP-dependent

29. Inhibitors of Protein Synthesis
Two important purposes to biochemists
These inhibitors have helped unravel the mechanism of protein synthesis
Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics
Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth
Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis

30. Protein synthesis inhibitors
Inhibitor Organism Mode of action
Initiation
Kasugamycin P Inhibits f-Met-tRNA binding
Streptomycin P Prevents initiation
Elongation (aminoacyltRNA binding)
Tetracycline P Inhibits aminoacyl-tRNA binding Streptomycin P Codon misreading
Elongation (peptide bond formation)
Chloramphenicol P Binds 50S, blocks peptidyl transf Cycloheximide E Inhib. transloc. of peptidyl-tRNA

31. Protein synthesis inhibitors
Inhibitor Organism Mode of action
Elongation (translocation)
Diphteria toxin E Inact. eIF2 thru ADP-ribosylati
Premature termination
Puromycin P, E Aminoacyl-tRNA analog
Acts as a peptidyl acceptor
Prevents further peptide elong.
Ribosome inactivation
Ricin E Catalytic inactiv. of 28S rRNA

32. Structures of common antibiotics

33. An NAD+-dependent ADP ribosylase
Diphtheria Toxin
An NAD+-dependent ADP ribosylase
One target of this enzyme is EF-2
EF-2 has a diphthamide
Toxin-mediated ADP-ribosylation of EF-2 allows it to bind GTP but makes it inactive in protein synthesis
One toxin molecule ADP-ribosylates many EF-2s, so just a little is lethal!

34. Diphtheria toxin - catalysis of NAD+-dependent ADP-ribosylation of proteins

35. from Ricinus communis (castor bean)
One of the most deadly substances known
A glycoprotein that is a disulfide-linked heterodimer of 30 kDa subunits
The B subunit is a lectin (a class of proteins that binds specifically to glycoproteins & glycolipids)
Endocytosis followed by disulfide reduction releases A subunit, which catalytically inactivates the large subunit of ribosomes

36. Ricin A subunit mechanism
Ricin A chain specifically attacks a single, highly conserved adenosine near position 4324 in eukaryotic 28S RNA
N-glycosidase activity of A chain removes the adenosine base
Removal of this A (without cleaving the RNA chain) inactivates the large subunit of the ribosome
One ricin molecules can inactivate 50,000 ribosomes, killing the eukaryotic cell!

37. Protein Folding
Translation is linked to transcription, proteins begin to fold while being synthesized by ribosomes
Proteins are assisted in folding by molecular chaperones - called chaperonins
Hsp60 and Hsp70 are two main classes
Hsp70 recognizes exposed, unfolded regions of new protein chains - especially hydrophobic regions
It binds to these regions, apparently protecting them until productive folding reactions can occur

38. Pathways of protein folding

39. The GroES-GroEL Complex
The principal chaperonin in E. coli
GroEL forms two stacked 7-membered rings of 60 kDa subunits; GroES is a dome on the top
Nascent protein apparently binds reversibly many times to the walls of the donut structure, each time driven by ATP hydrolysis, eventually adopting its folded structure, then being released from the GroES-GroEL complex
Rhodanese (as one example) requires hydrolysis of 130 ATP to reach fully folded state

40. GroEL-GroES complex
GroES
GroEL

41. Protein Translocation
An essential process for membrane proteins and secretory proteins
Such proteins are synthesized with a "leader peptide", a "signal sequence" of about amino acids
The signal sequence has a basic N-terminus, a central domain of 7-13 hydrophobic residues, and a nonhelical C-terminus
The signal sequence directs the newly synthesized protein to its proper destination

42. Protein Translocation
Four common features
Proteins are made as preproteins containing domains that act as sorting signals
Membranes involved in protein translocation have specific receptors on their cytosolic faces
Translocases catalyze the movement of the proteins across the membrane with metabolic energy (ATP, GTP, ion gradients) essential
Preproteins bind to chaperones to stay loosely folded

43. Prokaryotic Protein Transport
All non-cytoplasmic proteins must be translocated
The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding
The leader peptide also provides recognition signals for the translocation machinery
A leader peptidase removes the leader sequence when folding and targeting are assured

44. Eukaryotic Protein Sorting
Eukaryotic cells contain many membrane-bounded compartments
Most (but not all) targeting sequences are N-terminal, cleaveable presequences
Charge distribution, polarity and secondary structure of the signal sequence, rather than a particular sequence, appears to target to particular organelles and membranes
Synthesis of secretory and membrane proteins is coupled to translocation across ER membrane

45. Eukaryotic Signal Peptides
Postulated in 1970 by Hunter Blobel
(1999 Nobel prize)
N-terminal ER signal peptide (C-terminal ER-retention signal)
N-terminal mitochondrial signal peptide (chloroplast signals are similar)
Nuclear localization signal (not cleaved)

46. Events at the ER Membrane
As the signal sequence emerges from the ribosome, a signal recognition particle (SRP) finds it and escorts it to the ER membrane
There it docks with a docking protein or SRP receptor
SRP dissociates in a GTP-dependent process
Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved

47. Synthesis of a eukaryotic integral membrane protein and its translocation via the ER

48. Protein Degradation
Some protein degradation pathways are nonspecific - randomly cleaved proteins seem to be rapidly degraded
However, there is also a selective, ATP-dependent pathway for degradation - the ubiquitin-mediated pathway
Ubiquitin is a highly-conserved, 76 residue (8.5 kDa) protein found widely in eukaryotes
Proteins are committed to degradation by conjugation with ubiquitin

49. Ubiquitin and Degradation
Three proteins involved: E1, E2 and E3
E1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin
Ubiquitin is then transferred to a Cys-thiol of E2, the ubiquitin-carrier protein
Ligase (E3) selects proteins for degradation. the E2-S~ubiquitin complex transfers ubiquitin to these selected proteins
More than one ubiquitin may be attached to a protein target