Protein Synthesis Kim Foreman, PhD kforema@luc.edu Cancer Center, Room 235 August 10, 2018

Decoding mRNA ? mRNA proteins

Translation A A G U C A G U C Messenger RNA mRNA Codon Translation Polypeptide (amino acid sequence) Protein Lysine Serine Valine

The Genetic Code

The Genetic Code The genetic code is universal All known organisms use the same genetic code The genetic code is degenerate Multiple codons encode the same amino acid e.g. GGU GGC, GGA, and GGG all encode for glycine Degeneracy is mostly at the third base of the codon (Wobble base) Some codons have additional functions AUG encodes methionine Methionine can be used within a protein sequence and is generally the first amino acid at the beginning of the polypeptide chain UAA UAG and UGA do not encode for an amino acid. These are stop codons that terminate protein synthesis

An mRNA consisting of contiguous triplet codons can be read in three different reading frames.

Steps of Translation Charging of the tRNA Initiation Elongation Termination

Transfer RNA

Wobble permits some tRNAs to recognize more than one codon

Reactions catalyzed by aminoacyl-tRNA synthetases High energy ester linkage Eukaryotes: different synthetase of each amino acid Prokaryotes: one synthetase couples more than one amino acid

Reactions catalyzed by aminoacyl-tRNA synthetases

Interaction of a tRNA with its aminoacyl-tRNA synthetase Amino acid accepting arm ATP tRNA Aminoacyl- tRNA synthetase Anticodon region

Aminoacyl-tRNA synthetase must recognize the correct tRNA The anticodon is directly recognized by the synthetase through three adjacent binding pockets that are complementary to the shape and charge of the nucleotide in the anticodon. For amino acids with multiple unrelated anticodons, segments in the acceptor stem and elsewhere are critical. acceptor stem for the amino acid Site(s) in a tRNA used by one or more aminoacyl-tRNA synthetases to recognize their cognate tRNA. anticodon

Aminoacyl tRNA synthetase must select the correct amino acid correct amino acid has the highest affinity for the synthesis site proof-reading prevents the addition of the wrong amino acid. accuracy of tRNA charging is 1 mistake in 40,000 couplings

Translation initiation in prokaryotes Prokaryotic mRNA Initiation occurs at AUG codons with properly spaced Shine-Dalgarno sequence 5’ AUG SD AUG AUG SD AUG 3’ initiation codon with Shine-Dalgarno site internal Met codon does not have Shine-Dalgarno site initiation codon with Shine-Dalgarno site Shine-Dalgarno (SD) site consists of 3-9 contiguous bases in the mRNA that base pair with the 3’ end of 16S rRNA and is located ~5 bases upstream of the initiator codon

GAUUCCUAGGAGGUUUGACCUAUG CGA GCU The Shine-Dalgarno sequence functions as a binding site for the bacterial 16S ribosomal subunit 5’ 3’ AGCACGAGGGGAAAUCUGAUGGAACGCUAC E. coli trpA UUUGGAUGGAGUGAAACGAUGGCGAUUGCA E. coli araB GUAAACCAGGUAACAACCAUGCGAGUGUUG E. coli thrA CAAUUCAGGGUGGUGAAUGUGAAACCAGUA E. coli lacl AUGUACUAAGGAGGUUGUAUGGAACAACGC phage cro Pairs with Pairs with 16S rRNA initiator tRNA GAUUCCUAGGAGGUUUGACCUAUG CGA GCU 3’ 5’ UCCUCC mRNA U A C G 16S rRNA Shine-Dalgarno sequence

synthesis in prokaryotes Initiation of protein synthesis in prokaryotes The 16S rRNA subunit base pairs with the Shine-Dalgarno sequence immediately upstream from the initiating AUG codon. This positions the initiating codon in the ribosome. Because the ribosome assembles on the start codon, bacterial mRNA can be polycistronic. Translation initiation factors (IF1, IF2, IF3) orchestrate this interaction and assembly of the large ribosomal subunit to form the functional ribosome. Small ribosomal subunit provides the framework to accurately match the codon/anticodon while the large ribosomal subunit catalyzes the peptide bond formation In prokaryotes, translation begins with a special tRNA that carries a modified methionine- formylmethionine.

synthesis in eukaryotes Initiation of protein synthesis in eukaryotes Initiation tRNA (with bound eIF-2-GTP) is loaded into the small ribosomal subunit. eIF-4E and eIF-4G bind to both the 5’ cap and the poly A tail of the mRNA to ensure it is intact. The small ribosomal subunit binds to the mRNA by recognizing the cap and its associated eIFs. The small ribosomal subunit scans for the first AUG. eIF-2-GTP is hydrolyzed, eIFs dissociate and the large ribosomal subunit binds to the small subunit. The initiation tRNA is in the P-site of the ribosome. Protein synthesis is ready to begin.

Reactivation of eIF2-GDP eIF2+GTP eIF2-GDP during met-tRNA transfer eIF2-GDP must be recycled to eIF2-GTP . Exchange is catalyzed by eIF2B (guanine nucleotide exchange factor) Reactivation of eIF2 is catalyzed by eIF2B eIF2-GDP + GTP  eIF2-GTP + GDP

synthesis in eukaryotes Elongation of protein synthesis in eukaryotes Ribosome contains four binding sites mRNA site, A-site, P-site, E-site At the end of initiation, the initiation tRNA is in the P-site of the ribosome. A tRNA enters the A-site and base pairs with the codon of the mRNA. A peptide bond is formed between the amino acids in the A and the P-site. The peptidyl transferase catalytic activity of the large ribosomal subunit is accompanied by conformational changes that shift the two tRNAs into the E- and P- sites. Additional conformational changes move the mRNA 3 nucleotides so the ribosome is reset and ready for the next tRNA.

Elongation requires GTP hydrolysis Two elongation factors (EF-Tu and EF-G in bacteria; EF-1, EF-2 in eukaryotic cells). EF-Tu+GTP binds to a charged tRNA as it enters the ribosome. GTP hydrolysis and the exit of the EF-Tu from the ribosome cause two short lags that provide an opportunity for an incorrectly bound tRNA to exit the ribosome prior to incorporation into the polypeptide chain (responsible for 99.9% accuracy of the ribosome in translation) EF-G+GTP binds near the A-site and accelerates the movement of tRNAs to the P- and E-sites. The GTP hydrolysis catalyzes the conformational change that is needed to move the tRNA and advance the mRNA forward by one codon.

Termination of translation When a stop codon is reached, release factors bind to the ribosome and forces the peptidyl transferase of the ribosome to catalyze the addition of a water molecule instead of an amino acid. This frees the carboxyl terminus from the tRNA and the completed protein is released. Nakamura and ITO 2003, TIBS 28:99

Fidelity of protein synthesis (1) Aminoacyl tRNA synthetase must recognize the correct tRNA (2) Aminoacyl tRNA synthetase must select the correct amino acid (3) mRNA must be fully processed (in eukaryotes) prior to translation initiation (4) The ribosome matches the mRNA codon to the tRNA anticodon. The correct anticodon forms a stronger interaction with the codon than an incorrect pairing. (5) GTP hydrolysis and release of EF-Tu elongation factor provide short delays allowing the tRNA to be released from the A-site of the ribosome before an incorrect amino acid is irreversibly added into the peptide chain.

Differences in transcription and translation between prokaryotes and eukaryotes

Differences in transcription and translation between prokaryotes and eukaryotes

Comparison of a prokaryotic and eukaryotic ribosome

Puromycin – mimics tRNA “molecular mimicry”

Puromycin – an example of molecular mimicry

Creating a functional protein Folding Binding required co-factors Covalent modifications Assemble with any partner proteins

Proteins can begin to fold as they are synthesized

Molecular chaperones can assist in protein folding hsp70 Interact with exposed hydrophobic patches on proteins immediately after leaving the ribsome. ATP hydrolysis causes the hsp70 to bind tightly to the protein. Repeated binding and release help protein refold. hsp60 Forms a barrel shaped structure to isolate misfolded proteins. This prevents their aggregation and provides a favorable environment for them to refold.