Protein Biosynthesis: The Overall Picture

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1 Protein Biosynthesis: The Overall Picture

2 From: Biochemistry Berg, Tymoczko, Stryer 5 th Ed.

3 The Nature of the Genetic Code Three bases code for one amino acid WHY? Consider 4 n then 1 base can only code for 4 1 or 4 amino acids and 2 bases can only code for 4 2 or 16 amino acids but 3 bases can code for 4 3 = 64 amino acids From: Gamov 1954 Nature Vol. 173

4 The Nature of the Genetic Code Three bases code for one amino acid WHY? Consider 4 n then 1 base can only code for 4 1 or 4 amino acids and 2 bases can only code for 4 2 or 16 amino acids but 3 bases can code for 4 3 = 64 amino acids The code is not overlapping For example: CMB621andNGJarefun CMB 621 and NGJ are fun C MB6 21a ndngja ref un CM B62 1an dng Jar efu n

5 The Nature of the Genetic Code Three bases code for one amino acid WHY? Consider 4 n then 1 base can only code for 4 1 or 4 amino acids and 2 bases can only code for 4 2 or 16 amino acids but 3 bases can code for 4 3 = 64 amino acids 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

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7 Elliot "Ken" Volkin and Lazarus Astrachan discovered a DNA-like RNA in 1956 Ken Volkin Volkin infected bacterial cells of Escherichia coli with a bacteriophage virus, added phosphorus-32, isolated nucleic acid from the preparation, and hydrolyzed it with sodium hydroxide to make alkaline products that were separated using ionexchange chromatography. The results of experiments with phosphorus-32 were confirmed using a carbon-14 precursor that was specifically incorporated into the nucleic acid bases. Larry Astrachan joined Volkin in performing these experiments, which led to the discovery of messenger RNA, but they called it "DNA-like RNA." Lazarus Astrachan

8 Salvatore Luria, who became a Nobel laureate, convinced Volkin and Astrachan to publish their first paper on RNA research in the Journal of Virology in The paper announcing the discovery of a new kind of RNA is titled "Phosphorus Incorporation in E. Coli Ribonucleic Acid After Infection." In a book review in a 2001 issue of Nature, Horace Judson, a renowned historian of science who contributed to Time magazine, attributed the discovery of messenger RNA to François Jacob, Sydney Brenner, and Matthew Meselson. Weinberg published a letter in the November 29, 2001, issue of Nature disputing this claim. "In fact," he writes, "Jacob, Brenner, and Francis Crick, at an informal meeting on Good Friday 1960, suddenly 'discovered' the unique RNA found first in 1956 by Elliot Volkin and Lazarus Astrachan. Good accounts of this event can be found in The Statue Within by Jacob and What Mad Pursuit by Crick.

9 Translating the Message How does the sequence of mrna translate into the sequence of a protein? How do you translate the four-letter code of mrna into the 20-letter code of proteins? What are the mechanics like? There is no obvious chemical affinity between the purine and pyrimidine bases and amino acids As a way out of this dilemma, in 1955 Crick proposed adapter molecules these turned out to be transfer RNA or trna

10 Paul Zamecnik and Mahlon Hoagland discovered transfer RNA in 1956 In 1952, Zamecnik made a cellfree extract from rat liver with which he was able to synthesize proteins from amino acids. In 1953, using this system, Zamecnik and Hoagland showed that amino acids had to be energized, "activated," by ATP before they were incorporated into a peptide chain. Then in 1956, Hoagland followed up on an observation Zamecnik made earlier. Zamecnik noticed that low molecular weight RNA in the cell-free extract could be associated with radiolabeled amino acids. This led to the identification of trna the adaptors Francis Crick predicted in his Central Dogma.

11 The sequence and structure alanine trna was elucidated by Robert W. Holley in 1964

12 Biochemists Break the Code In 1961 Marshall Nirenberg and Heinrich Matthaei developed a cell-free system for protein synthesis and showed that poly-u produced polyphenylalanine Proceedings of the National Academy of Sciences of the United States of America, Vol. 47, No. 10. (Oct. 15, 1961), pp

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14 This work was quickly followed up by: Poly-A gave polylysine AAA - lysine Poly-C gave polyproline CCC - proline Poly-G gave polyglycine GGG - glycine But what about the others?

15 Getting at the Rest of the Code Work with nucleotide copolymers revealed some of the codes (H. Gobind Khorana)

16 Proceedings of the National Academy of Sciences 48, (1962):

17 Actual notes by Nirenberg

18 But Nirenberg and Leder cracked the entire code in 1964 They showed that trinucleotides bound to ribosomes could direct the binding of specific aminoacyl-trnas By using 14 C labeled amino acids with all the possible trinucleotide codes, they elucidated all 64 correspondences in the code

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20 The Nobel Prize in Physiology or Medicine 1968 "for their interpretation of the genetic code and its function in protein synthesis" Robert W. Holley H. Gobind Khorana Marshall W. Nirenberg

21 Features of the Genetic Code v All the codons have meaning 61 specify amino acids and the other 3 are stop codons v The code is unambiguous only one amino acid is indicated by each of the 61 codons v The code is degenerate except for Trp (W) and Met (M) each amino acid is coded by two or more codons v Codons representing the same or similar amino acids are similar in sequence v 2 nd base pyrimidine usually nonpolar amino acid v 2 nd base purine usually polar or charged amino acid

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23 The genomes of mitochondria, some prokaryotes and lower eukaryotes show some exceptions to the standard genetic code Some UGA codons in prokaryotes and eukaryotes (including humans) are used to specify selenocysteine an analog of cysteine

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26 Yeast alanine trna Structure (trna Ala )

27 The Wobble Hypothesis A single trna anticodon can recognize more than one, but not necessarily every, codon corresponding to a given amino acid This broader recognition can occur because of nonstandard pairing between bases in the so-called wobble position the third base in a mrna codon and the corresponding first base in its trna codon Note that there is a kinetic advantage to wobble: if all 3 base pairs in the codon-anticodon complex were strong Watson-Crick type then the interaction would be stronger and the trnas would dissociate more slowly from the mrna, slowing the rate of protein biosynthesis

28 Diseases associated with mutations in mitochondrial trna Over 150 mutations with documented pathogenicity have been identified within the human mitochondrial genome. MELAS - Mitochondrial Encephalomyopathy; Lactic Acidosis; Stroke Associated with mutations in mitochondrial trna leu A3243G mutation: 80% of MELAS syndromes Most affected individuals experience stroke-like episodes beginning before age 40. These mutations impair the ability of mitochondria to make proteins, use oxygen, and produce energy. Over 85% of people that carry this mutation present symptoms of diabetes: 75% of these patients develop hearing loss. There is no known treatment for the underlying disease, which is progressive and fatal.

29 Diseases associated with mutations in mitochondrial trna Over 150 mutations with documented pathogenicity have been identified within the human mitochondrial genome. Other MELAS mutation loci: T3271C (later onset age) Other syndromes with trna Leu mutations Riboflavin sensitive myopathy (T3250C) Isolated cardiomyophaty (A3243G; A3260G) MERRF Myoclonic Epilepsy with Ragged Red Fibers trna Lys : A8344G (Frequent); T8356C; G8363A In certain groups of stroke patients such as those younger than age 50 years, the prevalence of mitochondrial disease has been reported to be as high as 22%.

30 Activation of Amino Acids The Second Genetic Code The Aminoacyl-tRNA Synthetases must discriminate between 20 amino acids and many trnas (30-40 in bacteria and in eukaryotes) and link the right amino acid with the cognate trna This reaction serves two purposes: 1. It activates the amino acid so that it can readily react to form a peptide bond 2. It bridges the information gap between amino acids and codons

31 The Overall Aminoacyl-tRNA Synthetase Reaction

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33 The Two Classes of Aminoacyl-tRNA Synthetases Class I enzymes are usually monomeric and first add the amino acid to the 2 - OH before shifting it to the 3 -OH Class II enzymes are always oligomeric and add the amino acid directly to the 3 -OH Class I (a) and class II (b) aminoacyl-trna synthetases

34 Class I (left) and class II (right) aminoacyl-trna synthetases

35 Some Aminoacyl-tRNA Synthetases Have Proofreading Activity Isoleucine and valine are chemically similar and can both be accomodated in the active site of isoleucyl-trna synthetase as shown below The valyl-adenylate intermediate is formed about 1% of the time but the actually error rate for misincorporation of valine in place of isoleucine is only 1/10,000 This low rate is due to an ATP dependent proofreading step specifically, isoleucyltrna synthetase will catalyze the hydrolysis of the valyl-adenylate bond much of the time The total cost of proofreading all aminoacylations is estimated to be ~2% of the energy required to synthesize a bacterial cell

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37 Importance of Accuracy in the AminoacyltRNA Synthetase Reaction Once an aminoacyl-trna has been synthesized the amino acid part makes no contribution to accurate translation of the mrna Experiment by Seymour Benzer in 1963

38 Identity Elements Each trna molecule is recognized by a specific aminoacyl-trna synthetase Surprisingly, most recognition features are not limited to the anticodon in some cases they do not even include the anticodon Major identity elements in four trna species This insight was largely due to Paul Schimmel s research in the late 1980 s

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40 Alternative Decoding of UGA as Selenocysteine Several dozen proteins contain selenocysteine. Selenoprotein P is an abundant extracellular glycoprotein containing 7-10 selenocysteines. Selenoprotein mrnas contain a selenocysteine insertion sequence (SECIS) defined by sequence-specific and structure-specific properties This SECIS sequence inhibits termination

41 Also, a unique selenocysteyl-trna with an anticodon complementary to UGA is required trnasec has unusual features compared to other trnas. It is thought that these features prevent it interacting with the predominant EFTu. a 6 bp D-loop stem with a 4 bp loop; an extended acceptor stem-t stem axis of 13 bp a large variable loop. trna Sec is first charged with serine The trna-bound seryl residue is then converted to a selenocysteyl-residue by the enzyme selenocysteine synthase. The resulting Sec-tRNA(Sec) is specifically bound to an alternative translational elongation factor which delivers it to the ribosomes

42 From: Low and Berry, Trends Biochem Sci :203

43 Ribosomes were first observed in the mid-1950s by George Palade in the electron microscope as dense particles or granules. The term ribosome was proposed by Richard B. Roberts in 1958:

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47 30S 50S

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49 A closer look at the prokaryote ribosome

50 A Closer Look at the 30S Subunit

51 Ramakrishnan (2002) Cell Vol. 108

52 Secondary structure of E. coli 16S rrna

53 Assembly Map for 30S Subunit Masayasu Nomura This map shows how to reassemble the 30S subunit in the test tube from RNA and proteins The order of addition is important some proteins must be added before others will fit properly The abbreviation RNP stands for ribonucleoprotein particle

54 X-Ray Structure of the 50S Subunit RNA is shown in gray protein backbones are in gold. From: The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4A Resolution by Ban et al., (2000) Science 289:905

55 Tertiary and Secondary Structure of RNA in the 50S Subunit

56 The structure of the intact 70S ribosome has also been solved, although at lower resolution From: Science 2001 Vol. 292: Yusupov et al. Crystal Structure of the Ribosome at 5.5A Resolution

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58 Although the the X-ray structure of the eukaryotic 80S ribosome has not been solved yet there are cryoelectron microscope structures at intermediate resolution (11.7Å) as shown below 11.7Å resolution cryo-em map of the yeast 80S eef2 sordarin complex. The cryo-em map is shown (A) from the side; (B) from the top ; (C) from the 60S side, with 60S removed; and (D) from the 40S side, with 40S removed. From: EMBO J (2004) Vol. 23:1008 Spahn et al.

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61 Mechanics of Protein Biosynthesis All protein biosynthesis involves three phases initiation, elongation and termination Initiation involves binding of mrna and initiator aminoacyl-trna to the small ribosomal subunit, followed by binding of the large subunit Elongation: synthesis of all peptide binds with trnas bound to acceptor (A) and peptidyl (P) sites Termination occurs when a stop codon is reached

62 Prokaryotic Initiation The initiator trna is one with a formylated methionine: f-met-trna f Met It is only used for initiation regular Met-tRNA m Met is used for Met addition N-formyl methionine is the first amino acids of all E. coli proteins initially but is cleaved off in about half of the final products

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64 Shine-Dalgarno Sequence (John Shine and Lynn Dalgarno ) Correct registration of mrna on the ribosome requires alignment of a pyrimidine-rich sequence on the 3 -end of the 16S rrna with a purine-rich part of the 5 -end of the mrna The purine-rich sequence of the mrna is known as the Shine-Dalgarno sequence - some of these are shown below

65 Overview of Prokaryotic Initiation Initiation requires three proteins known as initiation factors (IFs) IF-1 and IF-3 bind to the 30S ribosome subunit IF-2 binds initiator trna and GTP mrna binds to the 30S ribosome along with the IFs, trna and GTP to form the 30S initiation complex GTP hydrolysis is accompanied by release of IF-1 and IF-2 and binding of the 50S subunit The N-formyl-met-tRNA Met f binds in the P or peptidyl site of the ribosome the E or exit site and the A or aminoacyl site are empty

66 The Prokaryotic Elongation Cycle Peptide chain elongation requires a set of proteins known as elongation factors these are elongation factor Tu (EF-Tu), elongation factor Ts (EF-Ts) and elongation factor G (EF-G) EF-Tu is the most abundant protein in E. coli it accounts for about 5% of the total protein

67 More on Elongation EF-Tu binds aminoacyl-trna and GTP forming the so-called ternary complex - binding of the aminoacylated trna to EF- Tu protects the labile ester bond from hydrolysis The ternary complex transports the aminoacyl-trna to the A site of the ribosome When the aa-trna is properly positioned, GTP is hydroyzed and EF- Tu-GDP dissociates from the ribosome EF-Ts recycles EF-Tu by exchanging GTP for GDP

68 EF-Tu-GDP is recycled to EF-Tu-GTP using EF-Ts

69 Prokaryotic Chain Elongation Role of EF-Tu-GTP

70 A special Elongation Factor - SelB - is required for selenocysteine :

71 Kinetic Proofreading Elongation Factor Tu carries out an important proofreading step which relies on the rate of GTP hydrolysis Consider the GTP hydrolysis rate in the following complexes: Complex EF-Tu-GTP EF-Tu-GTP-aatRNA (non-cognate) EF-Tu-GTP-aatRNA (cognate) GTPase rate (halflife) ~20,000 sec ~ 500 sec ~ 0.8 sec Hence, the GTP hydrolysis rate is greatly accelerated when the correct aatrna interacts with the mrna

72 How Accurate Must Protein Synthesis Be? The probability, p, of forming a protein with no errors depends on n, the number of amino acid residues and e, the frequency of inserting the wrong amino acid: p = (1 - e) n Observed values of e are close to 10-4

73 The Peptide Bond

74 Peptide Bond Formation This step is catalyzed by peptidyltransferase It is believed that this step involves a switch from the simple P and A states to hybrid states as indicated above

75 Peptidyltransferase The Ribosome is a Ribozyme! The high resolution structural studies of the 50S ribosome subunit also provided evidence that the peptidyltransferase activity of the ribosome does not directly involve proteins Hence, the 23S RNA in the large ribosomal subunit is a catalytic RNA or a ribozyme The paper by Nissen et al., (2000) Science 289:920 shows that there are no protein sidechains atoms closer than about 18 angstroms to the peptide bind being synthesized Their proposed mechanism is shown to the right

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77 PNAS August

78 Translocation Translocation in prokaryotes requires the third elongation factor, EF-G In this process GTP is hydrolyzed, the peptidyl-trna is moved to the P site and the empty trna is ejected from the E site

79 There is a striking structural similarity between the three dimensional structures of EF- G (shown on the right) and the ternary complex EF-Tu-GTPaatRNA (shown on the left) even though their composition is entirely different Molecular Mimicry It is speculated that this similarity allows EF-G to move temporarily into the A site, facilitating the displacement of the peptidyltrna complex

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82 Prokaryotic Peptide Chain Termination Proteins known as release factors recognize the stop codon at the A site There are three release factors: RF1, RF2 and RF3 RF1 recognizes the UAG stop codon RF2 recognizes the UGA stop codon Both factors recognize the UAA stop codon RF3 is a GTP-binding protein

83 Once the release factors occupy the A site on the ribosome, the ribosomal Peptidyl Transferase catalyzes transfer of the peptidyl group to water (hydrolysis). RF-3 then facilitates the release of RF-1 or RF-2

84 It has been assumed that RF1 and RF2 actually occupy the A site and make contact only with the termination codon but this may not be the case There is some evidence that the terminator codon may interact with a specific sequence in the rrna and that the function of the release factors is more indirect More to the Story?

85 Ribosome recycling factor (RRF) After termination, a deacylated trna and the mrna are still bound to the ribosome. Ribosome recycling factor (RRF), along with EF-G, dissociates the postranslational complex into mrna, trna and the ribosome

86 The exact mechanism of ribosome release is still controversial Two models exist which differ in the details concerning the order of binding and release of various factors Controversy over the action of RRF and EF-G exists,and two models have emerged. In model 1, RRF and EF-G not only catalyze the dissociation (splitting) of 70S ribosomes into subunits but they also catalyze the release of mrna and trna. By contrast, in model 2, RRF and EF-G catalyze only the dissociation of the 70S ribosome into subunits. In this model, initiation factor 3 (IF3) is required for the release of trna, and then mrna is released spontaneously.

87 More molecular mimicry! RRF and trna RRF blue trna - red

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