1. Protein Synthesis

2. Translating the Message How does the sequence of mRNA translate into the sequence of a protein? What is the genetic code? How do you translate the "four-letter code" of mRNA into the "20-letter code" of proteins? And what are the mechanics like? There is no obvious chemical affinity between the purine and pyrimidine bases and the amino acids that make protein. As a "way out" of this dilemma, Crick proposed "adapter molecules" - they are tRNAs!

3. The Collinearity of Gene and Protein Structures Watson and Crick's structure for DNA, together with Sanger's demonstration that protein sequences were unique and specific, made it seem likely that DNA sequence specified protein sequence Yanofsky provided better evidence in 1964: he showed that the relative distances between mutations in DNA were proportional to the distances between amino acid substitutions in E. coli tryptophan synthase

4. Elucidating the Genetic Code How does DNA code for 20 different amino acids? 2 letter code would allow for only 16 possible combinations. 4 letter code would allow for 256 possible combinations. 3 letter code would allow for 64 different combinations Is the code overlapping? Is the code punctuated?

6. The Nature of the Genetic Code A group of three bases codes for one amino acid The code is not overlapping The base sequence is read from a fixed starting point, with no punctuation The code is degenerate (in most cases, each amino acid can be designated by any of several triplets)

7. How the code was broken Assignment of "codons" to their respective amino acids was achieved by in vitro biochemistry Marshall Nirenberg and Heinrich Matthaei showed that poly-U produced polyphenylalanine in a cell-free solution from E. coli Poly-A gave polylysine Poly-C gave polyproline Poly-G gave polyglycine But what of others?

8. Getting at the Rest of the Code Work with nucleotide copolymers (poly (A,C), etc.), revealed some of the codes But Marshall Nirenberg and Philip Leder cracked the entire code in 1964 They showed that trinucleotides bound to ribosomes could direct the binding of specific aminoacyl-tRNAs By using C-14 labelled amino acids with all the possible trinucleotide codes, they elucidated all 64 correspondences in the code

10. Features of the Genetic Code All the codons have meaning: 61 specify amino acids, and the other 3 are "nonsense" or "stop" codons The code is unambiguous - only one amino acid is indicated by each of the 61 codons The code is degenerate - except for Trp and Met, each amino acid is coded by two or more codons First 2 codons of triplet are often enough to specify amino acid. Third position differs Codons representing the same or similar amino acids are similar in sequence (Glu and Asp)

12. tRNAs tRNAs are interpreters of the genetic code Length = 73 – 95 bases Have extensive 2 o structure Acceptor arm – position where amino acid attached Anticodon – complementary to mRNA Several covalently modified bases Gray bases are conserved between tRNAs

13. tRNAs: 2 o vs 3 o Structure

14. Third-Base Degeneracy Codon-anticodon pairing is the crucial feature of the "reading of the code" But what accounts for "degeneracy": are there 61 different anticodons, or can you get by with fewer than 61, due to lack of specificity at the third position? Crick's Wobble Hypothesis argues for the second possibility - the first base of the anticodon (which matches the 3rd base of the codon) is referred to as the "wobble position"

15. The Wobble Hypothesis The first two bases of the codon make normal H-bond pairs with the 2nd and 3rd bases of the anticodon At the remaining position, less stringent rules apply and non-canonical pairing may occur The rules: first base U can recognize A or G, first base G can recognize U or C, and first base I can recognize U, C or A (I comes from deamination of A) Advantage of wobble: dissociation of tRNA from mRNA is faster and protein synthesis too

18. AA Activation for Prot. Synth. Codons are recognized by aminoacyl-tRNAs Base pairing must allow the tRNA to bring its particular amino acid to the ribosome But aminoacyl-tRNAs do something else: activate the amino acid for transfer to peptide Aminoacyl-tRNA synthetases do the critical job - linking the right amino acid with "cognate" tRNA Two levels of specificity - one in forming the aminoacyl adenylate and one in linking to tRNA

19. Aminoacyl-tRNA Synthetase Amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi Most species have at least 20 different aminoacyl- tRNA synthetases. Typically one enzyme is able to recognize multiple anticodons coding for a single amino acids (I.e serine 6 different anticodons and only one synthetase) Two step process: 1)Activation of amino acid to aminoacyladenylate 2)Formation of amino-acyl-tRNA

20. Aminoacyladenylate Formation

21. Aminoacyl-tRNA Synthetase Rxn

23. Specificity of Aminoacyl- tRNA Synthetases Anticodon and structure features of acceptor arm of specific tRNAs are important in enzyme recognition Synthetases are highly specific for substrates, but Ile-tRNA synthetase has 1% error rate. Sometimes incorporates Val. Ile-tRNA has proof reading function. Has deacylase activity that "edits" and hydrolyzes misacylated aminoacyl-tRNAs

24. 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.

25. 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

26. 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

27. 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.

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

29. 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.

30. 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

31. 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.

32. 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

33. 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

34. 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.

35. 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.

36. 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.

37. 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

38. Peptide Bond formation

39. 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

40. 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.

41. 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

42. 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

43. 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

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

45. 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

46. 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

47. 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