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

2. Translating the Genetic Message
Protein biosynthesis is a complex process requiring ribosomes, mRNA, tRNA, and protein factors
Several steps are involved
Before being incorporated into growing protein chain, a.a. must be activated by tRNA and aminoacyl-tRNA synthetases

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

4. The Genetic Code
The ribosome moves along the mRNA three bases at a time rather than one or two at a time
Theoretically possible genetic codes are shown in figure Examples of Nonoverlapping and Continuous codes

5. The Genetic Code All 64 codons have assigned meanings
61 code for amino acids
3 (UAA, UAG, and UGA) serve as termination signals
only Trp and Met have one codon each

6. The Genetic Code
The third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg
Amino acids coded by 2, 3, or 4 triplets - the third letter of the codon - Gly, for example, is coded by GGA, GGG, GGC, and GGU

7. Wobble Base Pairing
Some tRNAs bond to one codon exclusively, but many tRNAs can recognize more than one codon because of variations in allowed patterns of hydrogen bonding
the variation is called “wobble”
wobble is in the first base of the anticodon

8. Base Pairing Combination in the Wobble Scheme

9. If there are 64 codons, how can there be less than 64 tRNA molecules?
The wobble hypothesis provides insight
in many cases, the degenerate codons for a given amino acid differ only in the third 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 misreading of the code; for example, if the Leu codon CUU were misread as CUC or CUA or CUG during transcription of mRNA, the codon would still be translated as Leu during protein synthesis
into some aspects of the degeneracy of the code

10. Amino Acid Activation
Amino acid activation and formation of the aminoacyl-tRNA take place in two separate steps
Both catalyzed by aminoacyl-tRNA synthetase
Free energy of hydrolysis of ATP provides energy for bond formation

11. tRNA Tertiary Structure
There are several recognition sites for various amino acids on the tRNA
there are specific binding sites on tRNAs that are recognized by an editing site in aminoacyl-tRNA synthetases

12. Chain Initiation
In all organisms, synthesis of polypeptide chain starts at the N-terminal end, and grows from N-terminus to C-terminus
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 initiation complex

13. The Initiation Complex

14. Chain Initiation tRNAmet and tRNAfmet contain the triplet 3’-UAC-5’
Triplet base pairs with 5’-AUG-3’ in mRNA
3’-UAC-5’ triplet on tRNAfmet recognizes the AUG triplet (the start signal) when it occurs at the beginning of the mRNA sequence that directs polypeptide synthesis
3’-UAC-5’ triplet on tRNAmet recognizes the AUG triplet when it is found in an internal position in the mRNA sequence
There are two different tRNAs for methionine in E.coli – one for unmodified methionine and one for N-formyl-methionine. The t-RNA forms are called met-tRNAmet and met-tRNAfmet (undergone formylation). In initiation process tRNA goes and positions itself onto mRNA

15. How does the ribosome know where to start translating
Start signal is preceded by a Shine-Dalgarno purine-rich leader segment, 5’-GGAGGU-3’
Lies about 10 nucleotides upstream of the AUG start signal and acts as a ribosomal binding site

16. Chain Elongation
Uses three binding sites for tRNA present on the 50S subunit of the 70S ribosome: P (peptidyl) site, A (aminoacyl) site, E (exit) site.
Requires
70S ribosome
codons of mRNA
aminoacyl-tRNAs
elongation factors EF-Tu (Elongation factor temperature-unstable), EF-Ts (Elongation factor temperature-stable), and EF-G (Elongation factor-GTP)
GTP, and Mg2+
P site binds to tRNA that carries a peptide chain, A site binds an incoming aminoacyl-tRNA and E site carries an uncharged tRNA that is about to be released from ribosome. EF-Tu guides aminoacyl-tRNA into part of A-site

17. Chain Elongation

18. Why is EF-Tu so important in E.coli?
Involved in translation fidelity
tRNA and aminoacid are mismatched then EF-Tu will not bind to t-RNA and will not deliver it to ribosome
Binds activated tRNA too well and does not release it from ribosome

19. Elongation Steps Step 1 Step 2 Step 3 Step 4
an aminoacyl-tRNA is bound to the A site
the P 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 peptide bond is formed, the P site is uncharged
Step 4
the uncharged tRNA is released
the peptidyl-tRNA is translocated to the P site
EF-G and GTP are required
the next aminoacyl-tRNA occupies the empty A site

20. 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

21. Chain Termination

22. Components of Protein Synthesis

23. Protein Synthesis
In prokaryotes, translation begins very soon after mRNA transcription
It is possible to have several molecules of RNA polymerase bound to a single DNA gene, each in a different stage of transcription
It is also possible to have several ribosomes bound to a single mRNA, each in a different stage of translation
Polysome: mRNA bound to several ribosomes
Coupled translation: the process in which a prokaryotic gene is being simultaneously transcribed and translated

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

25. Simultaneous Protein Synthesis on Polysomes

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

27. Eukaryotic Translation
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
stop codons are the same: UAG, UAA, and UGA
only one release factor that binds to all three stop codons

28. Posttranslational Modification
Newly synthesized polypeptides are frequently modified before they reach their final form where they exhibit biological activity
N-formylmethionine in prokaryotes is cleaved
leader sequences 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
disulfide 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

29. Examples of Posttranslational Modification
specific bonds in precursors are cleaved, as for example, preproinsulin to proinsulin to insulin

30. Protein Degradation Degradative pathways are restricted to
subcellular organelles such as lysosomes
macromolecular structures called proteosomes

31. Protein Degradation
In eukaryotes, ubiquitinylation (becoming bonded to ubiquitin) targets a protein for destruction
those with an N-terminus of Met, Ser, Ala, Thr, Val, Gly, and Cys are resistant
those with an N-terminus of Arg, Lys, His, Phe, Tyr, Trp, Leu, Asn, Gln, Asp, Glu have short half-lives
The nature of N-terminal amino acid influences its susceptibility to ubiquitinylation. Controls timing of destruction of a protein.

32. Acidic N-termini Induced Protein Degradation
Proteins with an acidic residue at N-terminus have a requirement for tRNA for its destruction. tRNA for arginine is used to transfer arginine to N-terminus making protein more susceptible to ubiquitin ligase.

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