Protein Translation

The genetic codeProtein synthesis Machinery

Ribosomes, tRNA’s

Universal translation: Protein Synthesis

DNA

mRNA

protein*

*ribosome

A,T,G,C

A,U,G,C

A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y

Chemical Letters

20 synthetases~45 tRNAs (E. coli)

1 tRNA <-> 1 amino acid

Amino Acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is activated for proteinsynthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of ATP hydrolysis is used to attach eachamino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxylgroup directly to an AMP moiety, forming and adenylated amino acid; the linkage of the AMP, normally an unfavorablereaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, theAMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3’ end of the tRNAmolecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNAmolecule. The synthetase enzyme is not shown in this diagram.

The genetic code is translated by means of two adaptors that act one afteranother. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular aminoacid to its corresponding tRNA molecule itself, whose anticodon forms base pairs with theappropriate codon on the mRNA. An error in either step would cause the wrong amino acid to beincorporated into a protein chain. In the sequence of events shown, the amino acid tryptophan (Trp)is selected by the codon UGG on the mRNA.

Molecular models of valine and isoleucine. The extra methylene group inisoleucine is marked. The synthetases specific for these amino acids are highlydiscerning.

Glutaminyl-tRNAsynthetase complex.The structure of thiscomplex reveals that thesynthetase interacts withbase pair G10:C25 inaddition to the acceptorstem and anticodon loop.

A comparison of the structure of procaryotic and eucaryotic ribosomes. Ribosomal components are commonly designatedby the their “S values,” which refer to their rate of sedimentation in an ultracentrifuge. Despite the differences in the numberand size of their rRNA and protein components, both procaryotic and eucaryotic ribosomes have nearly the same structure andthey function similarly. Although the 18S and 28S rRNAs of the eucaryotic ribosome contain many extra nucleotides notpresent in their bacterial counterparts, these nucleotides are present as multiple insertions that form extra domains and leave thebasic structure of each rRNA largely unchanged.

T.th. 70S5.5 Å

Head

Body

A sitetRNA

CP

L11

H69

30S 50S

Yusupov et al. (2001) Science 292, 883.h44

Formation ofthe initiationcomplex. Thecomplex forms in threesteps at the expense ofthe hydrolysis of GTPto GDP and Pi. IF-1, IF-2, and IF-3 areinitiation factors. Pdesignates the peptidylsite, A the aminoacylsite, and E, the exit site.Here the anticodon ofthe tRNA is oriented 3’to 5’, left to right.

Major Points1. Alternative RNA splicing: one mechanism evolved to expand diversity of gene products without increasing gene number2. Control of alternative splicing by positive and negative splicing factors ( analogous to transcriptional activators and repressors)3. Complexity of organisms reflected by exon numbers and differences between prokaryotic and eukaryotic mRNA’s4. Universal triplet codon converts NA seq into amino acid seq: total of 64 codes- AUG for Met and START, UAG,UAA&UGA for STOP and the rest for the remaining 19 amino acids5. Degeneracy of the code, ribosome entry site seq (Shine-Delgarno) anti-codon seq in tRNA6. High energy, amino-acyl-tRNA’s and specific synthetases7. Importance of adaptor molecules (ie. tRNA synthetases and anti-codon loop of tRNA’s)8. Complex structure of ribosomes: protein and RNA components

Formation of theinitiation complex.

The complex forms inthree steps at theexpense of thehydrolysis of GTP toGDP and Pi. IF-1, IF-2,and IF-3 are initiationfactors. P designates thepeptidyl site, A theaminoacyl site, and E,the exit site. Here theanticodon of the tRNAis oriented 3’ to 5’, leftto right.

Elongation cycle : binding of aminoacyl-tRNA, peptide-bond formation, and translocation.

First step inelongation(bacteria): binding ofthe secondaminoacyl-tRNA. Thesecond aminoacyl-tRNA entersthe A site of the ribosomebound to EF-Tu (shown here asTu), which also contains GTP.Binding of the secondaminoacyl-tRNA to the A site isaccompanied by hydrolysis ofthe GTP to GDP and Pi andreleaseof the EF-Tu•GDPcomplex from the ribosome.The bound GDP is releasedwhen the EF-Tu•GDP complexbinds to EF-Ts, and EF-Ts issubsequently released whenanother molecule of GTP bindsto EF-Tu. This recycles EF-Tuand makes it available to repeatthe cycle.

Second step inelongation(bacteria): formationof the first peptidebond. The peptidyltransferase catalyzing thisreaction is probably the 23SrRNA ribozyme. The N-formylmthionyl group istransferred to the amino groupof the second aminoacyl-tRNAin the A site, forming adipeptidyl-tRNA. At this stage,both tRNAs bound to theribosome shift position in the50S subunit to take up a hybridbinding state. The unchargedtRNA shifts so that its 3’ and 5’ends are in the E site. Similarly,the 3’ and 5’ ends of thepeptidyl tRNA shift to the Psite. The anticodons remain inthe A and P sites.

Third step inelongation(bacteria):translocation. Theribosome moves onecodon toward the 3’ endof mRNA, using energyprovided by hydrolysisof GTP bound to EF-G(translocase). Thedipeptidyl-tRNA is nowentirely in the P site,leaving the A site open ofthe incoming (third)aminoacyl-tRNA. Theuncharged tRNAdissociates from the Esite, and the elongationcycle begins again.

Termination ofprotein synthesis inbacteria. Terminationoccurs in response to atermination codon in the Asite. First, a release factor(RF1 or RF2 depending onwhich termination codon ispresent) binds to the A site.This leads to hydrolysis ofthe ester linkage betweenthe nascent polypeptide andthe tRNA in the P site andrelease of the completedpolypetide. Finally, themRNA, deacylated tRNA,and release factor leave theribosome, and the ribosomedissociates into its 30S and50S subunits.

A polyribosome. (A) Schematicdrawing showing how a series ofribosomes can simultaneously translatethe same eucaryotic mRNA molecule.(B) Electron micrograph of apolyribosome from a eucaryotic cell.

Ribosomes and endoplasmic reticulum. The electron micrograph and schematicdrawing of a portion of a pancreatic cell show ribosomes attached to the outer (cytosolic)face of the endoplasmic reticulum (ER). The ribosomes are the numerous small dotsbordering the parallel layers of membranes.

Major Points1. Multi-step protein synthesis : Initiation - small ribosome subunit loads onto mRNA at the entry site and (AUG) START codon along with IF1,2&3 followed by loading large subunit2. Three distinct binding pockets/sites in the ribosome complex: P- peptidyl ; A- aminoacyl ; and E - Exit sites3. Several steps during protein synthesis require hydrolysis of GTP to GDP & Pi (initiation complex formation; amino-acyl-tRNA binding and translocation)4. Peptidyl transferase reaction is catalyzed by ribozyme (RNA enzyme) within the small ribosomal subunit (23S or 30S)• Eukaryotic protein synthesis involves polyribosomes and a RNP complex in which the 5’ Cap of mRNA is linked to the 3’ poly A via various translation factors