1. From RNA to Protein
Lecture 6

2. Translation From RNA to Protein
Genetic code dictates how mRNA translated into aa sequence of protein
Nucleotides read consecutively in grps of 3 (codons)
Degenerate genetic code; 64 codons specify 20 aa
reading frames

3. Translation tRNA as an Adaptor Molecule
Recognizes and binds to codon and appr. aa
~80 nucleotdies
Folds into clover leaf and then into L-shaped structure
Two regions of unpaired nucelotdies at ends of L shaped molecule essential to function
1. anticodon= set of 3 consecutive nucleotides pairs w/complementary codon on mRNA
2. short ss region near 3’end where aa attaches

4. Translation Redundancy/Degeneracy of Genetic Code
More than one tRNA for many of the aa
Some tRNAs base pair w/ more than one aa; Wobble Position

5. Translation
tRNAs
Number and kinds of tRNAs varies across species ie: humans have 497 tRNA genes w/ only 48 diff anticodons represented
Transcribed by RNA Pol III
Some transcripts are spliced via cut-and-paste mechanism catalyzed by proteins (no lariat) occurs when tRNA is properly folded in cloverleaf conf
Extensive modifications > 50 different kinds
1 modification/10 nucleotides
Modifications essential to:
1) accuracy of tRNA attaching to correct aa
2) recognition of mRNA codon by tRNA anticodon

6. Translation Aminoacyl-tRNA Synthetases Couple aa to appr set of tRNAs
Aa specific
Enzymatic reaction attaches aa to 3’ end of tRNA requires ATP
High energy bond produced btwn aa and tRNA later used to covalently link aa to growing polypeptide chain

7. Translation Editing by tRNA synthetases
Selects correct aa in two step process:
1. correct aa highest affinity for active site
2. aa forced into second pocket whose
dimensions exclude correct aa
Those that enter second editing site
are hydrolyzed from AMP- hydrolytic editing
Raises overall accuracy of tRNA charging to
1 mistake/40,000 couplings
Synthetase must also recognize correct tRNAs

8. Translation
Ribosome Structure: rRNA molecules + 50 different proteins

9. Translation Ribosome Structure and Function
Large and Small Subunits; rRNA sequence highly conserved
66% RNA; 33% protein
rRNA responsible for:
structure
positioning tRNAs on mRNA
catalytic activity in forming peptide bond

10. Translation mRNA Decoded on Ribosomes
Ribosomal subunits assemble on mRNA near 5’ end
Ribosome translates mRNA in aa sequence using tRNAs as adaptors to add aa in correct sequence to end of growing polypeptide chain
aa linked by peptide bond formation
2 aa added/sec eucaryotic cell; 20 aa/sec added by bacteria
4 impt ribosomal binding sites

11. Translation Major Steps in Translation
Ribosomal Assembly and Initaition
Elongation
Termination and Release of Nascent Polypeptide

12. Translation Ribosomal Assembly
Initiation tRNA-Met & eIFs bind sm rRNA subunit
Sm rRNA subunit binds to 5’ end of mRNA recognizing CAP and 2 eIFs (eucaryotes)
Sm rRNA scans for AUG start
eIFs dissociate and lg rRNA binds
Initiator tRNA-met now in P-site leaving A-site vacant for incoming aminoacyl rRNA to start protein synthesis

13. Translation Procaryotes have Shine Delgarno Sequence
Specific sequence few nucleotides upstream of AUG start
AGGAGGU
Binding site positions sm rRNA subunit at AUG start
Bacterial mRNAs can be poly-cistronic; eucaryotes monocistronic

14. Translation Elongation
tRNA carrying next aa in chain binds to ribo A-site base pairing w/ mRNA codon
Peptide Bond Formation= carboxyl end released from tRNA at P-site and joined to free amino grp of aa linked to tRNA at A site forming new peptide bond
Translocation= conformational chgs move mRNA 3 nucleotides along ribo resetting ribo for next acyl tRNA

15. Translation Two Impt Elongation Factors Drive Translation
EF-Tu Mechanism- resp. 99% accuracy
a. Chged tRNA enters ribo bound to GTP form
b. Codon recognition triggers GTP hydrolysis
c. EF-Tu dissociates from ribo w/out tRNA
d. Introduces 2 short delays btwn codon anticodon pairing and chain elongation
EF-G binds in or near A site hydrolyzes GTP whose energy accelerates movement of bound tRNAs in A/P and P/E hybrid states

16. Translation Termination Stop codons: UAA, UAG, UGA
Stop codons recognized by “release factor” proteins
Release factors cause peptidyl transferase to catalyze addition of water to peptidyl tRNA
Hydrolysis frees COOH end of polypeptide chain from attachment to tRNA
Ribosomal dissassembly

17. Translation Proteins are Made on Polyribosomes
Syn of avg protein varies 20 sec to 2-3 min
mRNAs translated in form of polyribosomes w/ ribos spaced > 80 nucleotides
Transcription and translation occur simultaneously in procaryotes

18. Translation Quality Control of Translation Accuracy ~1 mistake/104 aa
Speed of translation 20 aa incorporated/sec in pro. vs 2 aa/sec euk.
Protein synthesis consumes more energy than any other biosynthetic process; 4 ATP equivalents/ aa
Quality control mech to ensure mRNA is complete recognition of Cap and poly A tail by initiation complex

19. Translation Antibiotics
Inhibitors of bacterial protein synthesis are effective antibiotics
Specificity of antibiotics useful for molecular cell biology studies

20. Life and Death of Proteins
Protein Maturation
Protein folding begins while protein is being synthesized
Maturation:
unique 3d structure
bind sm molecules
modifications
assemble w/ other proteins

21. Life and Death of Proteins
Protein Folding
Steps in protein folding:
1. molten globule
2. slow phase
Most of folding complete by time released from ribosome
Molecular chaperones held guide folding of many proteins

22. Life and Death of Proteins
Molecular Chaperonins
Two major families: hsp60 & hsp70
Affinity for exposed hydrophobic patches
Massage protein into folded conformation via ATP hydrolysis
Hsp70 acts early in life of protein; binds to stretch of 7 hydrophobic aa before protein leaves ribo
Hsp60 acts later in proteins life, forms barrel into which proteins fed

23. Life and Death of Proteins
Proteosome
Protein disposal apparatus dispersed throughout cytosolo
Hollow cylinder of proteases form stack of 4 heptameric rings
Ends composed of protein complex of ~20 polypeptides, 6 of which hydrolyze ATP to unfold proteins and move them into proteosome
Acts on proteins marked by ubiquitin

24. Life and Death of Proteins
Ubiquitin conjugating system
Multiubiquitin chain on target proteins recognized by receptors on proteosome
Targeted proteins: misfolded, oxidized, or other abnormal aa
Linear series of ubiquitin conjugates linked via lysine on target protein

25. Life and Death of Proteins
Regulated Destruction
Activation of compments in proteolytic pathway E1, E2, E3
Degradation signals created in response to intracellular or extracellular signals
All six pathways are used by cells to move protein into proteosome