1. Chapter 22 (Part 2) Protein Synthesis

2. Translation Slow rate of synthesis (18 amino acids per second) In bacteria translation and transcription are coupled. As soon as 5’ end of mRNA is synthesized translation begins. Situation in eukaryotes differs since transcription and translation occur in different cellular compartments.

3. Ribosomes Protein biosynthetic machinery Made of 2 subunits (bacterial 30S and 50S, Eukaryotes 40S and 60S) Intact ribosome referred to as 70S ribosome in Prokaryotes and 80S ribosome in Eukaryotes In bacteria, 20,000 ribosomes per cell, 20% of cell's mass. Mass of ribosomes is roughly 2/3 RNA

4. Prokaryotic Ribosome Structure E. coli ribosome is 25 nm diameter, 2520 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg 2+ 30S subunit is 930 kD with 21 proteins and a 16S rRNA 50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA

5. Eukaryotic Ribosome Structure Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar 40S subunit contains 30 proteins and 18S RNA. 60S subunit contains 40 proteins and 3 rRNAs.

6. Ribosome Assembly Assembly is coupled w/ transcription and pre-rRNA processing

7. Ribosome Structure Crystal structure of ribosome is known mRNA is associated with the 30S subunit Two tRNA binding sites (P and A sites) are located in the cavity formed by the association of the 2 subunits. The growing peptide chain threads through a “tunnel” that passes through the 40S (30S in bacteria) subunit.

8. Mechanics of Protein Synthesis All protein synthesis involves three phases: initiation, elongation, termination Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. Termination occurs when "stop codon" reached

9. Identification of Initiator Codon in Prokaryotes Involves binding of initiator tRNA (N- formylmethionyl-tRNA) to initiator codon (first AUG) The 30S subunit scans the mRNA for a specific sequence (Shine-Dalgarno Sequence) which is just upstream of the initiator codon. 16S RNA is involved in recognition of S-D sequence.

10. Prokaryotic Translational Initiation Formation of Initiation complex involves protein initiation factors IF-3 keeps ribosome subunits apart IF-2 identifies and binds initiator tRNA. IF-2 must bind GTP to bind tRNA. IF-1, IF-2, and IF-3 bind to 30S subunit to form initiation complex Once 50S subunit binds initiation complex, GTP is hydrolyzed, initiator tRNA enters P-site and IFs disassociate

11. Eukaryotic Initiation of Translation No S-D sequence. CAP binding protein (CBP) 5’ end of mRNA by binding to 5’ CAP structure An initiation complex forms with CBP, initiation factors and the 40S subunit. The complex then scans the mRNA looking for the first AUG closest to the 5’ end of the mRNA eIF-2 analogous to IF-2, transfers tRNA to P sight. GTP hydrolysis involed in release

12. Chain Elongation Three step process: 1)Position correct aminoacyl-tRNA at acceptor site 2)Formation of peptide bond between peptidyl-tRNA at P site with aminoacyl-tRNA at A site. 3)Shifting mRNA by one codon relative to ribosome.

13. Elongation Factor Tu (EF-Tu) binds to aminoacyl-tRNA and delivers it to the A site of the ribosome When EF-Tu binds GTP a conformational change occurs allowing it to bind to aminoacyl-tRNA.

14. EF-Tu-tRNA complex enters the ribosome and positions new tRNA at A site. If the anticodon matches the codon, GTP is hydrolyzed and EF-Tu releases the tRNA and then exits the ribosome.

15. Recycling of EF-Tu After leaving the ribosome EF-Tu- GDP complex associates with EF- Tscausing GDP to disassociate. When GTP bind to the EF-Tu/EF-Ts complex, EF-Ts disassociates and EF-Tu can bind another tRNA

16. Peptide Bond formation

17. Formation of Peptide Bond Once the peptide bond forms, the mRNA band shifts to move the new peptidyl-tRNA into the P-site and moves the deaminacyl-tRNA from the E-site Binding of EF-GTP to ribosome promotes the translocation Hydrolysis of EF-GTP to EF-GDP is required to release EF from ribosome and new cycle of elongation could occur

18. More on elongation Growing peptide chain then extends into the “tunnel” of the 50S subunit. Floding of the native protein does not occur until the peptide exits the “tunnel” Folding is facilitated by chaperones that are associated with the ribosome To ensure the correct tRNA enters the A site, the 16S RNA is involved in determing correct codon/anticodon pairing at positions 1 and 2 of the codon.

19. Eukaryotic elongation process Similar to what occurs in prokaryotes. Analogous elongation factors. EF-1a = EF-Tu  docks tRNA in A- site EF-1b = EF-Ts  recycles EF-Tu EF-2 = EF-G  involved in translocation process

20. Peptide Chain Termination Proteins known as "release factors" recognize the stop codon (UGA, UAG, or UAA) at the A site In E. coli RF-1 recognizes UAA and UAG, RF-2 recognizes UAA and UGA. RF-3 binds GTP and enhances activities of RF-1 and –2. 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 Hydrolysis of GTP is required for disassociation of RFs, ribosome subunit and new peptide

21. Protein Synthesis is Expensive! For each amino acid added to a polypeptide chain, 1 ATP and 3 GTPs are hydrolyzed. This is the release of more energy than is needed to form a peptide bond. Most of the energy is need to over-come entropy losses

22. Regulation of Gene Expression AAAAAA5’CAP mRNA RNA Processing RNA Degradation Protein Degradation Post-translational modification Active enzyme

23. Regulation of Protein Synthesis Regulation could occur at two levels in translation 1)Initiation – formation of the initiation complex 2)Elongation – elongation could be stalled by if an mRNA contains “rare” codons

24. Regulation of Globin gene translation by heme When heme is low, HCI kinase phosphorylates eIF-2- GDP complex, GEF binds tightly to phosphorylated eiF-2- GDP complex prevents recycling of eIF-2-GDP and stops translation

25. Regulation of the trp operon Transcription and translation are tightly coupled in E. coli. When Trp is aundant, transcription of the trp operon is repressed. The mechanism of this repression is related to translation of the