Translation Chapter 9

Overview Occurs on ribosomes-large aggregates of rRNA and protein tRNA acts as amino acid carriers Prokaryotes—occurs simultaneously with transcription and mRNA degradation Eukaryotes—occurs in cytoplasm mRNA translated 5’3’ Protein synthesis aminocarboxy

Protein Synthesis Polymerization of amino acids: condensation reaction (dehydration synthesis)

~Universal Genetic Code Codons—sets of 3 nucleotides corresponding to a single amino acid Each codon specifies a single amino acid More than one codon can specify the same amino acid code is said to be degenerate Some aa correspond to a single codon AUG—initiator codon, methionine (Met, M) UGG–Tryoptophan (TrP, W) Often codons encoding the same aa differ onl;y at the 3rd nucleotide Codons—sets of 3 nucleotides corresponding to a single amino acid Each codon specifies a single amino acid More than one codon can specify the same amino acid code is said to be degenerate Some aa correspond to a single codon AUG—initiator codon, methionine (Met, M) UGG–Tryoptophan (TrP, W) Often codons encoding the same aa differ onl;y at the 3rd nucleotide

~Universal Genetic Code The Genetic Code Chart shows aa assignments Non-random Tend to be clustered Reflects similar codons specifying the same aa Spontaneous mutations causing a single base change May not cause an aa change Similar aa are specified by similar codons Greatest similarities in first two nucleotides eg glycine – 4 codons – all GGX Greatest variability in third nucleotide of the triplet QUIZ—Codon for R, K, V, F aa for UCG, CCA, GGG

Why~Universal? Exceptions GUG sometimes used as a start Mammalian mitochondria Ciliated protozoa Selenocysteine NH3+ COO- H-C-CH2Se GUG sometimes used as a start (bacteria) Mammalian mitochondria- AUA start, other codons different than consensus. EX UGA-Trp (not Stop) AGG, AGA-Stop (not Arg) Ciliated protozoa—Ex UAA and UAG—Gln Selenocysteine An essential amino acid for selenoproteins EX. Glutathione oxidase Uses unique tRNA, initially bound to Ser Anticodon recognizes UGA (Stop) as Sel Signals in 3’ region determine Stop or Sel Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

Selenocysteine The 21st amino acid? An essential amino acid for selenoproteins EX. Glutathione oxidase Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt). Anticodon recognizes UGA (Stop) as Sec Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu) The 21st amino acid? An essential amino acid for selenoproteins EX. Glutathione oxidase Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt). Anticodon recognizes UGA (Stop) as Sec Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu) Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

Degeneracy—Wobble Hypothesis Explains how some tRNA recognize more than one codons tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide This third loose base pairing interaction is called wobble Note: only certain bases can substitute for others Explains how some tRNA recognize more than one codons tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide This third loose base pairing interaction is called wobble Note: only certain bases can substitute for others How? Canonical base pairing with the 1st two codon bases Loose, weak base pair interactions with the 3rd codon base #1 nucleotide of the anticodon is in a flexible domain of the tRNA Why? Kinetic advantage tRNA can dissociate more readily from the RNA template Allows faster protein synthesis

Wobble Example This UCA codon was read by the tRNA with a UGA anticodon But if this UCA was UCG, it would still have been read by the tRNA with a UGA anticodon In DNA , no pyr-pyr or pur-pur, but RNA is single stranded, allows more flexibility. Proposed by Crick (model building). Confirmed by experimentation. Implications for GE? Primer design

Codon Usage More than one codon exists for most amino acids (except Met and Trp) Organism may have a preferred codon for a particular amino acid Codon usage correlates with abundance of tRNAs (preferred codons are represented by abundant tRNAs) Rare tRNAs correspond to rarely used codons mRNAs containing rare codons experience slow translation Implications for GE?

Amino Acyl Synthetase and tRNA Amino acyl synthetases catalyze attachment of aa to its appropriate tRNA One for each amino acid tRNA Derived from large 1º transcript Heavily modified, unusual bases Extensive folding due to internal H-bonding There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids. It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code. The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases.

Amino Acyl Synthetase Carboxy end of aa attached to -phospate of ATP AMP released as carboxy end of amoinoacyl group transferred to O at C-3 of 3’nt When aa is attached, tRNA is charged or acylated No aa = uncharged Wrong aa = mischarged NOTE PPi Carboxy end of aa attached to -phospate of ATP AMP released as carboxy end of aa transferred to O at C-3 of 3’nt When aa is attached, tRNA is charged or acylated No aa = uncharged Wrong aa = mischarged NOTE PPi

tRNA Activation by Aminoacyl tRNA Synthetases 1. Aminoacyl-AMP formation: O HO (-)O R P O O O(-) P O O Adenine +H 3 N C P + PPi R O O O O O- Adenine O O(-) P +H 3 N C O O O- O OH OH Aminoacyl adenylate (Aminoacyl-AMP) 2Pi OH OH 2. Aminoacyl transfer to the appropriate tRNA: The generation of aminoacyl-tRNA's is a two-step process carried out by a family of enzymes called aminoacyl-tRNA synthetases. 1.The amino acid is first activated by conversion of the free carboxylic acid to aminoacyl adenlylate. Although stable, the aminoacyl-AMP intermediate does not leave the enzyme. 2. The amino acid is then transferred to the appropriate tRNA What happens to PPi? Because of the pyrophosphatases present in the cell, the pyrophosphate produce (Ppi, inorganic pyrophosphate), is quickly hydrolyzed to Pi, rendering the reaction thermodynamically favorable and irreverisble R R O O O Adenine +H 3 N C P + HO-ACC-tRNA +H 3 N C ACC-tRNA + AMP O O O- O O OH OH Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi

tRNA Function and Structure Anticodon-complementary to codon on mRNA Amino attachment (CCA) site Other recognition sites DHU loop TC Loop Extra arm (variable) NOTE: also unusual bases observed acceptor stem acceptor stem Anticodon-complementary to codon on mRNA. NOTE: no normal tRNA complementary to UGA, UAA, or UAG (except tRNSSec) Amino attachment (CCA) site. @ the 3’ end of the tRNA. This is the place at which the specific amino acid is attached These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G·U wobble base pair located at the third position within the acceptor helix of alanine tRNA.

tRNA Recognition by Amino Acyl Synthetase Sequence elements in each tRNA are recognized by its specific synthetase including: One or more bases in acceptor stem Base at position 73 “Discriminator base” Seems to play a major role in many cases, but in other cases it is completely ignored. In many, at least one anticodon base The features that permit recognition are not universal, yet there are some general rules. Recognition is not limited to the anticodon end of the tRNA. Discriminator base is invariant in every tRNA for a particular amino acid

No common set of rules for tRNA recognition !!! Recognition (cont’d) No common set of rules for tRNA recognition !!! Anticodon region is not the only recognition site The "inside of the L" and other regions of the tRNA molecule are also important Specificity of several aminoacyl-tRNA synthetases determined by: one or more bases in anticodon one or more bases in the acceptor stem discriminator base 73

Mischarging Observation: several aa similar in size and shape, but mischarging rare. Editing carried out by aminoacyl tRNA synthetase Ex Double sieve of isoleucine synthetase Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit. Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed). Sites can also distinguish based on hydrophobicity There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids. It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code. The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases. Observation: several aa similar in size and shape, but mischarging rare. Editing carried out by aa tRNA synthetase Ex Double sieve of isoleucine synthetase Expect 1 mischarge/225 events. Consequence? 1 misincorporated aa every time Ile is indicated. Intolerable. Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit. Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed). Sites can also distinguish based on hydrophobicity

Isoleucil-tRNA Synthetase: Proofreading Based on Size Recognition of aa by tRNA synthetases is based on size and hydrophilicity of aa chainThe recognition of amino acids by aminoacyl-tRNA synthetases is generally based on the size of the amino acid side chain.  •Acylation site rejects amino acids that are larger than the correct one because the binding site is too small. •Hydrolytic site destroys activated intermediates that are smaller than the right amino acid.   Example: Valine vs isoleucine (isoleucine has an extra methyl group) If valine is mistakenly activated by tRNA coding for isoleucine, it is hydrolyzed preventing its incorporation in tRNAIle : Acylation site rejects aa that are too large to fit in active site. Hydrolytric site destroys activated intermedeated that are smaller than correct aa Many synthases possses hydrolytic site. If Val is mistakenly activated the Ile tRNA, it will be hydrolyzed by smaller hydrolytic site Larger Acylation Site Smaller Hydrolytic Site Larger Acylation Site Smaller Hydrolytic Site CH 3 CH 3 H 3 C CH 3 O O +H 3 N NH 3 + O tRNAIle O tRNAIle CH 3 H 3 C CH 3 CH 3 O O +H 3 N +H 3 N O tRNAIle O tRNAIle Ile Val Misacylation Correct Acylation

Valyl tRNAVal Synthetase Proofreading: Hydrophobic/Polar Recognition Motif Acylation and hydrolytic sites can also discriminate based on hydrophobic verses polar interactions. Example: Valine vs threonine (difference is –OH in place of CH3) If threonine is mistakenly activated by tRNA coding for valine, it is hydrolyzed preventing its incorporation in tRNAVal :  Anticodon does not always participate in recognition Mutational studies have shown that recognition features are relatively simple Ale – GC pair in tRNA acceptor stem Aminoacyl-tRNA Synthetases Have two roles Aminoacyl-tRNA synthetases do the critical job - linking the right amino acid with "cognate" tRNA They act as a “scaffold” to match up the tRNA with its correct (“cognate”) amino acid They catalyze a two-step reaction This generates an ester linkage between -the 3’OH of the tRNA (on the acceptor stem) -and the COO- group of the amino acid2) This reaction activates the amino acid for protein synthesisAll members of one set of tRNAs for a particular amino acid (isoacceptor tRNAs) are served by one Aa tRNA synthetase.   Hydrophobic Acylation Site Polar Hydrolytic Site Hydrophobic Acylation Site Polar Hydrolytic Site 3 HC CH 3 H 3 C OH O O +H 3 N NH 3 + O tRNAVal tRNAVal O Difference in Hydrophobicity CH3 CH 3 HO CH 3 O O +H 3 N +H 3 N O tRNAVal O tRNAVal Val Thr Correct Acylation Misacylation

Experiment (1962) tRNA-ACA Protein has Cys Cys-tRNA-ACA Anticodon (recognizes UGU codon, encodes Cys) tRNA-ACA Cell-free extract amino acids & enymes tRNA is charged with Cys Protein has Cys RNA template UGUGUGUGUG... Cys-tRNA-ACA Treat w metal catalyst removes thiol groups Charged amino acid is changed chemically This experiment indicates the amino acid makes no contribution to accurate translation Protein has Ala RNA template UGUGUGUGUG... Ala-tRNA-ACA Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.

Protein Synthesis-3 Stages Initiation Elongation Termination

Ribosomes Composition of eukaryotic and prokaryotic ribosomes subunits, each composed of about 60% RNA and about 40% protein (by mass). Small ribosomal subunit 16S rRNA (bacteria), 18S rRNA (eukaryotes) 21 (bacteria) to 33 (eukaryote) proteins Initial binding of mRNA and initiator met-tRNA Large ribosomal subunit 23S & 5S rRNA (bacteria), 28S, 5.8S, & 5S rRNA (eukaryotes) 31 (bacteria) to 49 (eukaryote) proteins Forms complete, catalytic ribosome. Mol. Biol. Gene, Fig. 14-13

Composition of the E. coli Ribosome 50S subunit 23S & 5S RNA + 34 proteins 30S subunit 16S RNA + 21 proteins

Gross anatomy of the E. coli ribosome. head platform stalk Central protuberance platform ridge stalk Fig. 19.5

Initiation In both prokaryotes and eukaryotes, protein synthesis begins with a specific initiating tRNA In prokaryotes, initiator methionine amino group is methylated Attached to special tRNA (fMet-tRNA) Transformylase—adds formyl group Deformylase—removes formyl group from Met of completed peptide Formylation does not occur in eukaryotes

} Steps in Initiation Association of 30S subunit with mRNA fMet-tRNA Initiation factors (3 proteins) GTP QUESTION: AUG encodes fMet and Met. How does 30S ribosome “know” which aa is to be inserted? } 30S Pre-initiation Complex

Initiator Codon Recognition fMet-tRNA responds only to initiator codons (AUG, GUG, UUG [rarely]) Met-tRNA responds only to internal AUG Meaning of codons dependent on their context, i.e., sequences nearby In Eukaryotes: 5’ cap involvement In Prokaryotes: Shine-Dalgarno Sequence mRNA- (5’)AGGAG (3’) 16S rRNA- (3’)UCCUC(5’) fMet-tRNA responds only to initiator codons (AUG, GUG, UUG [rarely]) Met-tRNA responds only to internal AUG Meaning of codons dependent on their context, i.e., sequences nearby In Euks: 5’ cap involvement In Proks: Shine-Dalgarno Sequence mRNA- (5’)AGGAGG(3’) 16S rRNA- (3’)UCCUCC(5’)

Shine-Dalgarno Interaction Upstream from initiator AUG Complementary to a stretch on 16S rRNA Seen in virtually all prokaryotic mRNA

Initiation Factors in Protein Synthesis IF-1 Promotes dissociation of ribosome. IF-1 also blocks the A site of the small ribosomal subunit  insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation. IF-2 small GTP-binding protein (a GTPase). Interacts with Small subunit IF1 fMet-tRNAfMet IF-1 Promotes dissociation of ribosome. IF-1 also blocks the A site of the small ribosomal subunit  insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation. IF-2 small GTP-binding protein (a GTPase). Interacts with: Small subunit IF1 fMet-tRNAfMet

Initiation Factors in Protein Synthesis IF-2 (cont’d) IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit IF-3 Binds small subunit, prevents reassociation w/ large subunit binds mRNA to the 30S ribosomal subunit frees it from its complex with the 50S subunit. IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit IF-3 Binds small subunit, prevents reassociation w/ large subunit binds mRNA to the 30S ribosomal subunit frees it from its complex with the 50S subunit. As mRNA binds, IF-3 helps to correctly position the complex such that the tRNAfMet interacts via base pairing with the mRNA initiation codon (AUG).

30S Pre-initiation Complex IF-1 Promotes dissociation of ribosome. IF-1 also blocks the A site of the small ribosomal subunit  insures the initiation aa-tRNA fMet-tRNAfMet can bind only in the P site & that no other aa-tRNA can bind in the A site during initiation. IF-2 small GTP-binding protein (a GTPase). Interacts with Small subunit IF1 fMet-tRNAfMet IF-2/GTP helps the initiator helps it dock with the small ribosome subunit, prevents other tRNAs from binding small subunit IF-3 Binds small subunit, prevents reassociation w/ large subunit binds mRNA to the 30S ribosomal subunit frees it from its complex with the 50S subunit.

70S Initiation Complex Assembly 30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, fMet-tRNAfMet When fMet-tRNAfMet pairs with initiator codon, small subunit undergoes conformational change Result: Release of IF-3 Large subunit can bind small subunit complex Binding of large subunit stimulates GTPAse activity of IF-2/GTP hydrolysis of GTP to GDP IF-2/GDP and IF-1 fall off RESULT: 70S ribosome with fMet-tRNAfMet in P-site of ribosome As the large ribosomal subunit joins the complex, GTP on IF-2 is hydrolyzed, leading to dissociation of IF-2-GDP and dissociation of IF-1. A domain of the large ribosomal subunit serves as GAP (GTPase activating protein) for IF-2. Once the two ribosomal subunits come together, the mRNA is threaded through a curved channel that wraps around the "neck" region of the small subunit..

RESULT: 70S Initiation Complex

Overview Dissociation of inactive 70S IF-1, IF-3 IF-2/tRNA, mRNA fMet-tRNAfMet/initiator codon-releases IF-3 GTP Hydrolysis, releases IF-1, IF-2 Complete 70S complex

70S Ribosome A (aminoacyl) site Small subunit Transfer RNAs P (peptidyl) site Large subunit (50 S) 5’ end Messenger RNA

Alternative View A=Aminoacyl site P=peptidyl site E=Exit site

Elongation Overview Aminoacyl tRNA complementary to codon in A-site moves into A-site N-formyl-Met transferred from tRNAfMet to aminoacyl-tRNA in A-site now have a dipeptide in the A-site tRNAfMet leaves P-site Ribosome moves along mRNA (translocation) dipeptide in P-site New aminoacyl-tRNA moves into A site, etc INSERT

ELONGATION INSERT Elongation CHAIN ELONGATION: 3-stage elongation rxn cycle Rate Up to 40 residues/ sec Requires non-ribosomal proteins (elongation factors) and a ribozyme (part of 23S rRNA) INSERT

Transpeptidation Reaction Carboxy end of nascent peptide Nucleophilic attack A ring nitrogen (N) of the catalytic adenosine (at the CCA end of tRNA) may promote the reaction by extracting a H+ from the attacking amino N. This H+ is then donated to the hydroxyl of the tRNA in the P site, as the ester linkage is cleaved. Amino terminus of incoming amino acid

Transpeptidation Completed The nascent polypeptide, one residue longer, is now linked to the tRNA in the A site. However, translocation has already partly occurred, because peptide bond formation is associated with rotation of the single-stranded 3' end of the A-site tRNA toward the P-site, positioning the aminoacyl moiety for catalysis. This rotary movement also positions the nascent polypeptide to feed into the entrance to the protein exit tunnel, which is located midway between A & P sites.

EF-G-GTP Role in translocation Binding site uncovered EF-G-GTP occupies Hydrolysis GDP leaves open A-site Role in translocation. After translocation, shift in A-site tRNA uncovers a binding site for EF-g-GTP in large subunit portion of A-site.

Translation Termination “Stop” Codon No anticodons, but are release factors Proteins Occupy A-site Activate hydrolysis of peptide from peptidyl-tRNA

Translation Termination 2 Release factors RF-1 and RF-2 recognize stop codons RF-3 –stimulates dissociation of 70S ribosome after release of polypeptide chain Anticodon recognition determined 3 aa Release factors RF-1 and RF-2 recognize stop codons RF-I UAG RF-2 UGA Both --UAA RF-3 –stimulates dissociation of from ribosome after release of polypeptide chain Anticodon recognition determined 3 aa in RF-3 (a peptide anticodon).

Proofreading in Translation Codon:anticodon base pairing 16S rRNA forms H-bonds with minor groove of codon:anticodon duplex only when correctly paired

3. Proofreading in Translation Correct base pairing allows EF-Tu bound to aa-tRNA to interact with factor binding center, inducing GTP hydrolysis and EF-Tu release Incorrect base pairing FBC not contacted allows more time for EF-Tu GTP release Correct base pairing allows EF-Tu bound to aa-tRNA to interact with factor binding center, inducing GTP hydrolysis and EF-Tu release Incorrect base pairing allows more time for EF-Tu GTP release Pausing caused by activation allows non-cognate tRNA to dissociate. It leaves before EF-Tu not incorporated.

4. Proofreading in Translation Incorrectly paired tRNA can’t rotate into position for peptide bond formation “tRNA accommodation” Only correctly base-paired aa-tRNA remain associated w/ribosome as they rotate into the correct position for peptide bond formation. Rotation referred to as tRNA accomodation.

Antibiotics Translation the target of many antibiotics. EX Site of Nucleophilic attack Absent terminates translation Puromycin

Eukaryotic Translation Factors designated with the prefix "e" EF-Ts replaced by eEF-1 eEF-2 (target for diphtheria toxin) Eukaryotic Translation Factors designated with the prefix "e" EF-Ts replaced by eEF-1 eEF-2 NOTE: eEF-2 is a specific target for diphtheria toxin (catalyzes addition of ADP to eEF-2) Toxin acts catalytically Eventually kills cell by halting protein synthesis a few µg sufficient to kill an unimmunized individual Formerly a leading cause of childhood death EF2 is only known substrate for diphtheria toxin EF2 contains rare modification of one of histidine residues and this is site recognized by toxin Mutant cells that cannot modify site are resistant Addition of ADP-ribose inactivates EF2 Kills cells by irreversible block of protein synthesis COMPOSED OF 2 PARTS: B-fragment—binds receptor A-fragment—business end, inserted by B-fragment Therapeutic potential

Diphtheria Toxin EF2 is only known substrate for diphtheria toxin EF2 contains rare modification of one of histidine residues and this is site recognized by toxin Mutant cells that cannot modify site are resistant Addition of ADP-ribose inactivates EF2 Kills cells by irreversible block of protein synthesis P. aeruginosa exotoxin A works same as diphtheria toxin