The Mechanism of Translation

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Allison Fisher
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1 The Mechanism of Translation

2 The central dogma Francis Crick 1956 pathway for flow of genetic information Transcription Translation Duplication DNA RNA Protein 1954 Zamecnik developed the first cell-free system (rat liver extract) to perform protein synthesis Crick postulated adaptor hypothesis that the amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part that actually fits onto the RNA template Zamecnik and Hoagland discovered aminoacyl synthetases, enzymes responsible for attaching amino acids to trnas before incorporation into proteins By 1961 it was known that polypeptide chains were synthesized on ribosomes, and that different trna molecules carry the appropriate amino acid to the ribosome depending on the genetic codon on the mrna.

3 Complexity of translation [amounts in one E. coli cell] Translation machinery is 35% of dry weight of cell 20,000 ribosomes 200,000 trnas 100,000 proteins and cofactors

4 Summary of Protein Synthesis aa1 + trna1 aminoacyl-trna synthetase aa1-trna1 ATP AMP + PPi aa1-trna1 + aa2-trna2 peptidyl transferase on ribosome aa1-aa2-trna2 + trna1 (released) aa3-trna3 + aa1-aa2-trna2 peptidyl transferase on ribosome aa1-aa2-aa3-trna3 + trna2 (released)

5 transcription codon translation growing peptide chain amino acid incoming trna

6 The machinery responsible for translating the language of mrnas into the language of proteins is composed of four primary components: mrna provides the information for translation. The protein-coding region of the mrna consists of an ordered series of three nucleotide- long units called codon that specify the order of amino acids. trna provides the physical interface between the amino acids being added to the growing polypeptide chain and the codons in the mrna. Aminoacyl-tRNA synthetases couples amino acids to specific trnas that recognize the appropriate codon(s). Ribosome coordinates recognition of the mrna by each trna and catalyzes peptide-bond formation between the growing polypeptide chain and the amino acid attached to the selected trna.

7 What is the organization of nucleotide sequence information in mrna?

8 Organization of nucleotide sequence information in mrna The protein-coding region(s) of each mrna is composed of a contiguous, non-overlapping string of codons called an OPEN READING FRAME (ORF). Each ORF specifies a single protein and starts and ends at internal sites within the mrna. The first and last codons of an ORF are known as the START and STOP CODONS.

9 Organization of nucleotide sequence information in mrna START CODON In bacteria, the start codon is usually 5 -AUG-3, but 5 -GUG-3 and sometimes even 5 -UUG-3 are also used. Eukaryotic cells always use 5 -AUG-3 as the start codon. The start codon has two important functions: First, it specifies the first amino acid to be incorporated into the growing polypeptide chain. Second, it defines the reading frame for all subsequent codons.

10 Organization of nucleotide sequence information in mrna STOP CODON Stop codons, of which there are three (5 -UAG-3, 5 -UGA-3, and 5 -UAA-3 ), define the end of the ORF and signal termination of polypeptide synthesis. MONOCISTRONIC AND POLYCISTRONIC mrna Eukaryotic mrnas almost always contain a single ORF (monocistronic mrnas). In contrast, prokaryotic mrnas frequently contain two or more ORFs and consequently can encode multiple polypeptide chains (polycistronic mrnas).

Prokaryotic mrnas Have a Ribosome-Binding Site That Recruits the

upstream (on the 5 side) of the start codon called the RIBOSOME-BINDING

located 3 9 bp on the 5 side of the start codon, is complementary to a

11 Prokaryotic mrnas Have a Ribosome-Binding Site That Recruits the Translational Machinery Many prokaryotic ORFs contain a short sequence upstream (on the 5 side) of the start codon called the RIBOSOME-BINDING SITE (RBS) or SHINE-DALGARNO SEQUENCE (5 -GGAGG-3 ) The RBS, typically located 3 9 bp on the 5 side of the start codon, is complementary to a sequence located near the 3 end of one of the ribosomal RNA components, the 16S ribosomal RNA (rrna)

12 Eukaryotic mrnas Are Modified at Their 5 and 3 Ends to Facilitate Translation Eukaryotic mrnas recruit ribosomes using a specific chemical modification called the 5 -CAP, which is located at the extreme 5 end of the mrna. Two other features of eukaryotic mrnas stimulate translation. One feature is the presence, in some mrnas, of a purine three bases upstream of the start codon and a guanine immediately downstream (5 - G/ANNAUGG-3, KOZAK SEQUENCE). These bases are thought to interact with the initiator trna, not with an RNA component of the ribosome as in prokaryotes)

Eukaryotic mrnas Are Modified at Their 5 and 3 Ends to Facilitate Translation A second feature that contributes to efficient translation is the presence of a poly-a tail at the extreme 3

13 Eukaryotic mrnas Are Modified at Their 5 and 3 Ends to Facilitate Translation A second feature that contributes to efficient translation is the presence of a poly-a tail at the extreme 3 end of the mrna. Despite its location at the 3 end of the mrna, the poly-a tail enhances the level of translation of the mrna by enhancing the recruitment of key translation initiation factors.

14 trna structure

15 trna loops functions Every trna has the sequence ACC on the 3 to which the amino acid is attached T-loop Involved in recognition by the ribosome. D loop recognition by the aminoacyl trna synthetases that charge the appropriate amino acid Anticodon loop base pairs with the mrna codon

form what appears as a continuous helix.

16 Two Coaxial stacked arms form the L-shape of trna Coaxial stacking, or helical stacking, occurs when the nucleotide bases from two separate base-paired stems stack and align to form what appears as a continuous helix. 7 bp acceptor stem in trna stacks on the 5 bp T stem to form an A- type helical arm. The D stem and anticodon stem stack to form a second helical arm

17 How do aminoacyl-trna synthetases recognize and attach the correct amino acids to each trna?

18 aminoacyl-trna charging Overall Overall reaction reaction amino acid + ATP + trna aminoacyl-trna + AMP + PPi 2-step reaction 1: amino acid is activated by adenylylation 2: amino acid is transferred to to the 3 or 2 OH of the ribose of the terminal A on trna

19 aminoacyl-trna charging Adenylylation of amino acid An O atom of the amino acid α-carboxyl attacks the P atom of the initial phosphate of ATP amino acid Transfer of AMP-high energy bond adenylylated amino acid pyrophosphate

20 aminoacyl-trna charging Product retains a high-energy bond joining the amino acid to the trna Unusual in that this is an ester linkage Provides the thermodynamic energy to drive protein synthesis Hydrolysis of PPi to 2 Pi can drive the synthesis of aminoacyl-trna Transfer of adenylylated amino acid to trna adenylylated amino acid the 2' or 3' OH of the terminal adenosine of trna attacks the amino acid carbonyl C atom high-energy bond

21 aminoacyl-trna charging Aminoacyl-tRNA synthetases 20 enzymes, 1 per amino acid One synthetase for each amino acid a single synthetase may recognize multiple trnas for the same amino acid Each aa-trna synthetase: must recognize several cognate trnas several or all the trnas whose anticodons complement the codons specifying a particular amino acid must recognize the correct amino acid Two different classes of aminoacyl-trna synthetases, based on 3D structure

22 aminoacyl-trna charging trna O (terminal 3 nucleotide of appropriate trna) There are 2 families of Aminoacyl-tRNA Synthetases: Class I & Class II. O P O O CH 2 H H O C O 3 2 O H H OH Adenine HC R NH 3 + Aminoacyl-tRNA Two different ancestral proteins evolved into the 2 classes of aars enzymes, which differ in the architecture of their active site domains. They bind to opposite sides of the trna acceptor stem, aminoacylating a different OH of the trna (2' or 3').

23 aminoacyl-trna charging Attach aminoacyl group to 2 hydroxyl group of 3 terminal nucleotide Mostly monomeric proteins Attach aminoacyl group to 3 hydroxyl group Mostly dimeric proteins I

to the acceptor stem and anticodon loop.

24 aminoacyl-trna charging Require anticodon for proper recognition Often recognize only features in the acceptor stem Synthetase interacts with base pair G10:C25 in addition to the acceptor stem and anticodon loop. Synthetase binds to both the acceptor stem and anticodon loop. Glutaminyl-tRNA Synthetase Complex Threonyl-tRNA Synthetase Complex

25 aminoacyl-trna charging acceptor discriminator stem base 3 acceptor end 5 anticodon stem anticodon loop Highly specific elements required for aa-trna synthetase recognition on BOTH business ends of the trna anticodon 3 Cocrystal structure of glutaminyl aminoacyl-trna

26 aminoacyl-trna charging Adenylylation of amino acid Transfer of adenylylated amino acid to trna

27 aminoacyl-trna charging

28 aminoacyl-trna charging Mechanism by the aminoacyl synthetase recognizes the correct amino acid 1. Enzyme preferentially binds the correct amino acid - each amino acid fits into an active site pocket in the enzyme - network of hydrogen bonds, electrostatic and hydrophobic interactions so that only amino acids with a sufficient number of favorable interactions will bind Tyrosine can be distinguished by the hydroxyl group Isoleucine is larger-excluded from valrs active site Extra CH 3 on Ile provide extra kcal/mol free energy 2. Selectively edits the the incorrect amino acid

29 aminoacyl-trna charging Hydrolytic editing Flexible CCA arm of trna moves amino acid between activation site and editing site. If amino acid fits well into editing site, it is hydrolyzed from the AMP (or from the trna if aminoacyl-trna already formed)

30 aminoacyl-trna charging The frequency of mischarging is very low; typically, less than 1 in 1000 trnas is charged with the incorrect amino acid If aminoacyl-trna synthetase makes a mistake, the wrong amino acid is incorporated. Ribosome cannot discriminate between correctly and incorrectly charged trnas

31 aminoacyl-trna charging cysteinyl-trna Cys was reacted with nickel hydride, which causes desulfurization of the aminoacyl moiety, to form alanly-trna Cys In cell-free protein synthesizing system, alanyl trna Cys introduces alanines at codons specifying cysteine The protein synthesizing machinery was unable to detect that the incorrect amino acid was attached to trna Cys

32 The ribosome

The small subunit contains the DECODING CENTER in which charged trnas read or decode the codon units of the mrna.

33 The Ribosome The ribosome is composed of two subassemblies of RNA and protein known as the large and small subunits. The large subunit contains the PEPTIDYL TRANSFERASE CENTER, which is responsible for the formation of peptide bonds. The small subunit contains the DECODING CENTER in which charged trnas read or decode the codon units of the mrna. By convention, the large and small subunits are named according to the velocity of their sedimentation when subjected to a centrifugal force

The Ribosome The unit used to measure sedimentation velocity is the SVEDBERG (S; the larger the S value the faster the sedimentation velocity and the larger the molecule) In bacteria, the large

34 The Ribosome The unit used to measure sedimentation velocity is the SVEDBERG (S; the larger the S value the faster the sedimentation velocity and the larger the molecule) In bacteria, the large subunit has a sedimentation velocity of 50 Svedberg units whereas the small subunit is called the 30S subunit. The intact prokaryotic ribosome is referred to as the 70S ribosome. Note that 70S is less than the sum of 50S and 30S! The explanation for this apparent discrepancy is that sedimentation velocity is determined by both shape and size and is not an exact measure of mass. The eukaryotic ribosome is somewhat larger, composed of 60S and 40S subunits, which together form an 80S ribosome.

35 The Ribosome Prokaryotic ribosomes 5S rrna (120 nucleotides) prokaryotic ribosome 23S rrna (2900 nucleotides) ~34 proteins 16S rrna (1540 nucleotides) 21 proteins

36 The Ribosome Eukaryotic ribosomes 5.8S rrna (160 nucleotides) eukaryotic ribosome 5S rrna (120 nucleotides) 28S rrna (4,700 nucleotides) 49 proteins 18S rrna (1,900 nucleotides) ~33 proteins

37 The Ribosome The base-paired structure of the Escherichia coli 16S rrna. Standard base pairs (G- C, A-U) are shown as bars; non-standard base pairs (e.g. G-U) are shown as dots. Secondary structure is more highly conserved than primary sequence, i.e. complementary mutations evolve to maintain base paring.

38 The Ribosome Evolutionarily distant 16S-like rrnas: predicted secondary structures archaebacteria yeast bovine mitochondria

39 The Ribosome Three-dimensional fold of rrna in 70S ribosome Yusupov et al. Science 292, 883 (2001)

40 The Ribosome The ribosomal proteins are important for maintaining the stability and integrity of the ribosome, but NOT for catalysis ie. the ribosomal RNA acts as a ribozyme Large subunit. Grey = rrna Gold = ribosomal proteins

41 The Ribosome Ribosomal proteins often have extensions that snake into the core of the rrna structure Crystal structure of L19 L15 (yellow) positioned in a fragment of the rrna

42 The Ribosome Two-dimensional gel electrophoretograms of E. coli 30S and 50S ribosomal proteins Kaltschmidt and Wittmann (1970)

43 The Ribosome 2D PAGE

44 The Ribosome Proteins are first separated by electrophoresis according to charge, then separated according to their size.

45 The Ribosome Experiments to elucidate ribosome structure Electron microscopy: combination of micrographs from one particle and/or oriented micrographs from different particles Sequencing of rrna and secondary structure prediction Antibody-tagged ribosomal proteins were localized by electron microscopy and neutron diffraction Crystallization and X-ray diffraction of ribosome and subunits A. Yonath, Weizman Institute of Science, Israel V. Ramakrishnan, MRC Lab of Molecular Biology, England H. Noller, University of California-Santa Cruz, USA T. Steitz, Yale University, USA

46 First Ribosome models obtained by Negative Staining A

47 First Ribosome models obtained by Negative Staining Lake, 1971

48 Immunoelectron Microscopy

49 Lake, 1971 Immunoelectron Microscopy

50 Transfer RNA in the ribosome CCA acceptor 50S Peptidyl transferase center 30S anticodon

The Ribosome The Ribosome Has Three Binding Sites for trna To perform the peptidyl transferase reaction, the ribosome must be able to bind at least two trnas simultaneously.

51 The Ribosome The Ribosome Has Three Binding Sites for trna To perform the peptidyl transferase reaction, the ribosome must be able to bind at least two trnas simultaneously. In fact, the ribosome contains three trna-binding sites, called the A-, P-, and E-sites. The A site is the binding site for the aminoacylated-trna, The P site is the binding site for the peptidyl-trna, and The E site is the binding site for the trna that is released after the growing polypeptide chain has been transferred to the aminoacyl-trna (E is for exiting ).

52 The Ribosome X-ray structure: trna binding sites peptidyl transferase center Ribosomal subunits in the X-ray structure of the T. thermophilus in complex with three trnas and an mrna. E: Exit site for free trna P: Peptidyl-tRNA A: Aminoacyl-tRNA Yusupov et al. Science 292, 883 (2001)

53 The Ribosome Interaction between A-site and P-site trnas and mrna Helps to maintain correct reading frame Kink between A- and P-site codons

54 The Ribosome 12 intersubunit contacts hold together the 70S ribosome RNA-RNA contacts protein-protein and protein-rna contacts

55 The Ribosome a tunnel through the large subunit that allows the growing polypeptide chain to pass out of the ribosome

56 The Ribosome

57 50S RNA Protein 30S RNA Protein peptidyl transferase center decoding center

58 Translation

59 Protein Synthesis

Initiation prokaryotes IF3 promotes the dissociation of the ribosome and occupies eventual E site IF1 binds near the A site and blocks it IF2(GTP) binds to IF1 and reaches

60 Initiation prokaryotes IF3 promotes the dissociation of the ribosome and occupies eventual E site IF1 binds near the A site and blocks it IF2(GTP) binds to IF1 and reaches over A site into P site and assists binding of fmet-trna RBS of mrna basepairs with 16S RNA Order of fmet-trna and mrna binding is random The 30S pre-initiation complex is formed

61 Initiation Codon-anticodon interaction occurs resulting in conformational changes in the small subunit 50S ribosomal subunit binds and stimulates GTP hydrolysis IF2(GDP) + IF1 dissociate from ribosome 70S initiation complex is formed

62 Prokaryotic mrnas are polycistronic Ribosome is recruited by Shine-Dalgarno sequence (Ribosome Binding Site (RBS)) Translation Initiation Region (TIR) 5-9 nt upstream of start codon Complementary to 3 end of 16S rrna Shine-Dalgarno sequence start start start UAA UAG UGA bases RBS start AUG AUGA translational coupling

63 Shine-Dalgarno sequence Base pairing between ribosome binding site in mrna and 3 end of 16S rrna Some translational initiation sequences recognized by E. coli ribosomes.

64 Shine-Dalgarno sequence recruits the ribosome to mrna

65 places the start codon in right position (P-site) in the ribosome Shine-Dalgarno sequence

fmet-trna trna f Met is structurally different from the "regular" trna Met fmet-trna has unique features that distinguish it as the initiator RNA

66 fmet-trna trna f Met is structurally different from the "regular" trna Met fmet-trna has unique features that distinguish it as the initiator RNA The terminal bases of the acceptor arm are not paired as they are in all other trnas. It contains 3 consecutive G C base pairs in the anticodon stem.

67 fmet-trna A single MetRS aminoacylates both trna Met and trna i Met trna f Met is functionally different from the "regular" trnamet in two important ways: It is recognized by a transformylase that will catalyse the formylation of methionyl-trna f Met. Transformylase catalyses the formylation of the methionyl-trna f Met but not of methionyl-trna Met or uncharged trna. The enzyme uses N10- formyltetrahydrofolate as the formyl group donor.

68 fmet-trna In bacteria, protein synthesis starts with a special amino acid: N-formyl-methionine a peptide bond The presence of the formyl group on the methionine after it has been attached to trna f Met serves two purposes: This will be the only charged trna that is positioned in the peptidyl (P) site on the ribosome It ensures that fmet-trna f Met will not be used for internal methionine codons

69 fmet-trna fmet-trna f Met H CH 3 -S-CH 2 CH 2 C-COO-tRNA N-H C=O H N-Terminal Block Blocking the N-terminal amino acid assures that the peptide chain will grow towards the C-terminal

70 Initiation Initiation factors guide and position ribosome

71 Initiation IF1 guides initiator trna to the P-site

72 Elongation aminoacyl-trna binding to A site peptide bond formation peptide bond translocation

75 accomodation of trna in PTC

76 In addition to codon-anticodon interaction, adenines in 16S rrna form tight interaction with minor groove of codon-anticodon base pair Correctly paired A:U and G:C similar in minor groove Correctly paired trnas have lower rate of dissociation from ribosome

released No GTP hydrolysis EF-Tu trna released Correct codon-anticodon base-pairing favors

77 correct pairing polypeptide chain factor binding center GTP in correct position for hydrolysis incorrect pairing factor binding center not contacted GTP hydrolysis and EF-TU released No GTP hydrolysis EF-Tu trna released Correct codon-anticodon base-pairing favors induction of GTP hydrolysis necessary for EF-Tu release GTPase is not activated if not positioned correctly

78 aa-trna must rotate into correct position for peptide bond formation accomodation correct base pairing incorrect base pairing

79 Catalysis requires 1-3 Å range 3 ends of A- and P-site trnas

80 Peptidyl transferase reaction amino group of aa-trna attacks carbonyl group of most carboxy-terminal amino acid of peptidyl-trna peptide bond formed growing polypeptide chain transferred from peptidyl-trna to aminoacyl-trna

81 Basepairing between 23S rrna and CCA ends of trnas in A and P sites position α-amino group to attack carbonyl group Proton shuttle 2 -OH of 3 A of peptidyl-trna donates H to 3 -OH and accepts proton from attacking α-amino of aa

factor binding center trna hybrid states 3

Codon-anticodon base pairing disrupted in E-

82 factor binding center trna hybrid states 3 ends in P-site, anticodon end in A-site (pre-peptidyl transferase position) Binding of EF-G-GTP stimulates GTP hydrolysis Codon-anticodon base pairing disrupted in E- site, trna dissociates EF-G changes conformation, triggers translocation

83 EF-Tu-GDPNP-Phe-tRNA EF-G.GDP Both bind to A site of decoding center

84 Exchanging GTP for GDP EF-Tu has a higher affinity for GDP than for GTP EF-TU GDP cannot bind aminoacylated trnas EF-Ts (a GTP exchange factor) binds to EF-Tu GDP and displaces GDP GTP binds to EF-Tu EF-Ts and displaces EF-Ts leaving EF-Tu - GTP

86 Release factors Class Class I I 3 amino acid (peptide) anticodon RF1: SPF peptide anticodon RF2: PAT peptide close anticodon to decoding center determine release-factor specificity in vivo Conserved region in peptidyl transferase site: GGQ directly or indirectly involved in polypeptide hydrolysis

87 Release factor 1 trna mimics trna RF1 bound to A-site

88 Peptide anticodon close to codon Conserved GGQ close to 3 end of P-site trna and peptidyl transferase center

89 peptide hydrolysis factor binding site

90 peptide hydrolysis Presence of Class 1 RF obligatory for RF3-GDP binding

91 Disassembly requires ribosomal recycling factor EF-G stimulates release of trnas in P and E sites RRF binds to empty A site trna mimic recruits EF-G

Chapter 32: Translation

Chapter 32: Translation Voet & Voet: Pages 1343-1385 (Parts of sections 1-3) Slide 1 Genetic code Translates the genetic information into functional proteins mrna is read in 5 to 3 direction Codons are