1. Chapter Twelve Protein Synthesis: Translation of the Genetic Message

2. Translating the Genetic Message
Protein biosynthesis is a complex process requiring _________________, _________________, & _________________
Several steps are involved
Before being incorporated into growing protein chain, a.a. must be _________ by tRNA and ___________________

3. The Genetic Code Salient features of the genetic code
___________: a sequence of three bases (a codon) is needed to specify one amino acid
_________________: no bases are shared between consecutive codons
___________: no intervening bases between codons
____________________: more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets
_______________: the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria

4. The Genetic Code (Cont’d)
The __________ moves along the mRNA ______ bases at a time rather than ________ at a time
Theoretically possible genetic codes are shown in figure 12.2

5. The Genetic Code (Cont’d)
All 64 codons have assigned meanings
61 code for ____________ _____________
3 (UAA, UAG, and UGA) serve as _____________ _____________ signals
only ______ and _______ have one codon each
the ______ ______ base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg
the second base is important for the ______ of amino acid; for example, if the second base is U, the amino acids coded for are hydrophobic
for the 15 amino acids coded for by 2, 3, or 4 triplets, only the _______ letter of the codon varies. For example, Gly is coded for by GGA, GGG, GGC, and GGU

6. The Genetic Code (Cont’d)

7. The Genetic Code (Cont’d)
Assignments of triplets in genetic code based on _________________________________________
______ mRNA: if mRNA is polyU, polyPhe is formed; if mRNA is poly --ACACACACACACACACACACA--, poly(Thr-His) is formed
_____________: aminoacyl-tRNAs bind to ribosomes in the presence of trinucleotides
synthesize ________________ by chemical means
carry out a ________________ for each type of trinucleotide
_______________-tRNAs are tested for their ability to bind in the presence of a given trinucleotide

8. The Filter-Binding Assay

9. Wobble Base Pairing
Some tRNAs bond to one codon exclusively, but many tRNAs can recognize more than one codon because of variations in ______________________
the variation is called “wobble”
wobble is in the _________ base of the _________

10. Base Pairing…Wobble Scheme

11. Wobble Base Pairing

12. Wobble Base Pairing Hypothesis
The wobble hypothesis provides insight into some aspects of the _________________ of the code
in many cases, the degenerate codons for a given amino acid differ only in the ______ base; therefore fewer different tRNAs are needed because a given tRNA can base-pair with several codons
the existence of wobble minimizes the damage that can be caused by a _______________ of the code; for example, if the Leu codon CUU were misread CUC or CUA or CUG during transcription of mRNA, the codon would still be translated as Leu during protein synthesis

13. Amino Acid Activation
Amino acid activation and formation of the aminoacyl-tRNA take place in two separate steps
Both catalyzed by amionacyl-tRNA _________________
Free energy of hydrolysis of _______ provides energy for __________________

14. Amino Acid Activation (Cont’d)
This two-stage reaction allows selectivity at two levels
______________: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site in the tRNA synthetase
________: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases.

15. tRNA Tertiary Structure
Recognition sites for various amino acids on the tRNA

16. Chain Initiation
In all organisms, synthesis of polypeptide chain starts at the ____________ end, and grows from _________________ to ________________
Initiation requires:
tRNAfmet
initiation codon (AUG) of mRNA
30S ribosomal subunit
50S ribosomal subunit
initiation factors IF-1, IF-2, and IF-3
GTP, Mg2+
Forms the _______________ _______________

17. The Initiation Complex

18. Chain Initiation
tRNAmet and tRNAfmet contain the triplet 3’-_______-5’
Triplet base pairs with 5’-AUG-3’ in mRNA
3’-UAC-5’ triplet on tRNAfmet recognizes the AUG triplet (start signal) when it is at the ____________ of the mRNA sequence that directs polypeptide synthesis
3’-UAC-5’ triplet on tRNAmet recognizes the AUG triplet when it is found in an _________________ _________________ in the mRNA sequence
Start signal is preceded by a Shine-Dalgarno purine-rich leader segment, 5’-GGAGGU-3’, which usually lies about ___ nucleotides _______________ of the AUG start signal and acts as a ________ binding site

19. Chain Elongation
Uses 3 binding sites for tRNA present on the 50S subunit of the 70S ribosome: P (___________) site, A (________) site, E (______) site
Requires
70S ribosome
codons of mRNA
aminoacyl-tRNAs
GTP, and Mg2+
elongation factors:
EF-Tu (Elongation factor temperature-unstable),
EF-Ts (Elongation factor temperature-stable), and
EF-G (Elongation factor-GTP)

20. Shine-Dalgarno Sequence Recognized by E. Coli Ribosomes

21. Elongation Steps Step 1 Step 2 Step 3 Step 4
an aminoacyl-tRNA is bound to the ___ site
the ___ site is already occupied
2nd amino acid bound to 70S initiation complex. Defined by the mRNA
Step 2
EF-Tu is released in a reaction requiring EF-Ts
Step 3
the _______ _______is formed, the P site is uncharged
Step 4
the uncharged tRNA is released
the _____________-tRNA is translocated to the P site
EF-G and GTP are required
the next aminoacyl-tRNA occupies the _________ ___ site

22. Chain Elongation

23. Chain Termination Chain termination requires
stop codons (UAA, UAG, or UGA) of mRNA
RF-1 (Release factor-1) which binds to UAA and UAG or RF-2 (Release factor-2) which binds to UAA and UGA
RF-3 which does not bind to any termination codon, but facilitates the binding of RF-1 and RF-2
GTP which is bound to RF-3
The entire complex dissociates, setting free the completed polypeptide, the release factors, tRNA, mRNA, and the 30S and 50S ribosomal subunits

24. Chain Termination

25. Components of Protein Synthesis

26. Protein Synthesis
In prokaryotes, translation begins very soon after mRNA transcription
Several molecules of RNA polymerase may be bound to a single DNA gene, each in a different stage of transcription
Several ribosomes may be bound to a single mRNA, each in a different stage of translation
_____________: mRNA bound to several ribosomes
_________________ _________________ : the process in which a prokaryotic gene is being simultaneously transcribed and translated

27. Simultaneous Protein Synthesis on Polysomes
A single mRNA molecule is translated by several ribosomes _________________
Each ribosome produces a copy of the polypeptide chain specified by the mRNA
When protein has been completed, the ribosome ____________________________ that are used again in protein synthesis

28. Simultaneous Protein Synthesis on Polysomes (Cont’d)

29. Eukaryotic Translation
Eukaryotic Chain Initiation:
• the most different from process in prokaryotes
• 13 more _________ __________are given the designation eIF (eukaryotic initiation factor) (Table 12.4)

30. Eukaryotic Translation (Cont’d)
Chain elongation
uses the same mechanism of peptidyl transferase and ribosome translocation as prokaryotes
there is no E site on eukaryotic ribosomes, only A and P sites
there are two elongation factors, eEF-1 and eEF-2
eEF2 is the counterpart to EF-G, which causes translocation
Chain termination
______ codons are the same: UAG, UAA, and UGA
only one _________ __________ binds to all three _____________ _____________

31. Posttranslational Modification
Newly synthesized polypeptides are frequently ___________ before they reach their final form where they exhibit biological activity
N-formylmethionine in prokaryotes is _______________
specific bonds in precursors are cleaved, as for example, preproinsulin to proinsulin to insulin
___________ _________are removed by specific proteases of the endoplasmic reticulum; the Golgi apparatus then directs the finished protein to its final destination
factors such as heme groups may be attached
___________ bonds may be formed
amino acids may be modified, as for example, conversion of proline to hydroxyproline
other covalent modifications; e.g., addition of carbohydrates

32. Examples of Posttranslational Modification

33. Protein Degradation
Proteins are in a _________ state and are often turned over
Degradative pathways are restricted to
subcellular organelles such as lysosomes
macromolecular structures called proteosomes
In eukaryotes, ____________ (becoming bonded to _______________) targets a protein for destruction
protein must have an N-terminus
those with an N-terminus of Met, Ser, Ala, Thr, Val, Gly, and Cys are ________________
those with an N-terminus of Arg, Lys, His, Phe, Tyr, Trp, Leu, Asn, Gln, Asp, Glu have ______ half-lives

34. Ubiquitin-Proteosome Degradation

35. Acidic N-termini Induced Protein Degradation