Protein Synthesis (Translation)

Proteins Synthesis Translation Process by which ribosomes convert the information carried by mRNA to synthesize new proteins mRNA is translated from 5 to 3-end producing a protein (peptide) synthesized from amino to carboxyl end

Requirements Ribosomes Amino acids mRNA tRNA Enzymes Protein factors (initiation, elongation & release factors) ATP & GTP as source of energy

Ribosome The ribosome has three binding sites for tRNA The P site The A site The E site

Schematic model showing binding sites on ribosome P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit

Mammalian example of 80S rRNA

Proteins Synthesis (Steps) I. Activation of amino acids (formation of amino-acyl-tRNAs or charged tRNA) Amino-acyl-tRNA-synthetases recognize and catalyze the covalent attachment of a specific amino acid to CCA arm of tRNA Amino acid + tRNA Amino-acyl-tRNA ATP AMP + PPi  2Pi First amino acid is always methionine (met-tRNA)

1. Amino acid Aminoacyl-tRNA synthetase (enzyme) Pyrophosphate Phosphates tRNA AMP Aminoacyl tRNA (an “activated amino acid”)

II. Codon-anticodon recognition Codon (on mRNA) & anti-codon (on tRNA) are anti-parallel to one-another

An initiation codon marks the start of an mRNA message AUG = methionine Start of genetic message End Figure 10.13A

Proteins Synthesis (Steps) III. Initiation Proper mRNA is selected by a ribosome ; ribosome attaches to it and reads the message from 5 to 3 end of mRNA Ribosome dissociates into 40s & 60s sub-units Formation of Pre-initiation complex (PIC) [40 s rRNA + met-tRNA + GTP + initiation factors (eIFs)] mRNA binds to PIC forming 43 s initiation complex

More Initiation The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.

Proteins Synthesis (Steps) Binding of 43s (IC) with 60s ribosome forms 80s initiation complex, this complex contains P, A & E sites (“P” for peptidyl tRNA , “A” for new amino-acyl-tRNA & “E” for exit) Starter / initiator codon (AUG) is identified by tRNA near the 5 end (cap end) of mRNA This is determined by other sequences ,“Kozak consensus sequences” that surround “AUG” met-tRNA occupies “P” site and is ready for elongation

mRNA, a specific tRNA, and the ribosome subunits assemble during initiation Large ribosomal subunit Initiator tRNA P site A site Start codon Small ribosomal subunit mRNA 1 2

Elongation A cyclic process; one amino acid is added at a time Sequence determined by the order of codons in mRNA Requires “Elongation factors” (EFs) Steps Binding of amino acyl-tRNA to the “A” site Peptide bond formation of this incoming amino acid with the carboxyl group (end) of met-tRNA (or growing peptide chain) at “P” site Catalyzed by “Peptidyl-transferase” – a component of 28s rRNA (ribozyme) of the 60s ribosomal subunit

Elongation This results in attachment of growing peptide to tRNA in the “A” site Translocation – Now the ribosome advances three nucleotides (1 codon) towards 3-end of mRNA; results in Movement of free tRNA (uncharged) from “P” to “E” site for release EF2 displaces peptidyl-tRNA from “A” to “P” site “A” site is now open for another amino-acyl-tRNA for another cycle of elongation

Termination Elongation continues until a termination codon is encountered Termination occurs when one of the three termination codons moves into “A” site These codons are recognized by release factors Release factors bind to and induce peptidyl transferase to release the polypeptide from tRNA

Termination Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon. When the ribosome reaches a stop codon, there is no tRNA that binds to it. Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate. The new polypeptide is completed. Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).

1. Recognition 3. Translocation 2. Peptide bond formation Amino end of polypeptide E 3¢ mRNA P site A site Ribosome ready for next aminoacyl tRNA 5¢ 2 GTP 2 GDP E E P A P A GDP GTP 3. Translocation 2. Peptide bond formation E P A

Ribosome translates 5’ to 3’ on mRNA. Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E tRNA mRNA 3¢ Codons 5¢ Schematic model with mRNA and tRNA Ribosome translates 5’ to 3’ on mRNA. Polypeptide chain grows amino end first, carboxyl end last.

3¢ 3¢ 5¢ Release factor Stop codon (UAG, UAA, or UGA) Free polypeptide When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. 3¢ The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. The two ribosomal subunits and the other components of the assembly dissociate.

Polyribosomes -a single mRNA (transcript) is translated by many ribosomes simultaneously mRNA+ bound ribosomes= polyribosomes or polysome Allows fast synthesis of many copies a polypeptide

Polyribosome or Polysome Completed polypeptides Growing polypeptides Incoming ribosomal subunits Polyribosome Start of mRNA (5¢ end) End of mRNA (3¢ end) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 mm This micrograph shows a large polyribosome in a prokaryotic cell (TEM).

RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 mm RNA polymerase DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5¢ end)

Targeting Polypeptides to Specific Locations In eukaryotes, what are the two populations of ribosomes? Free, soluble in cytosol synthesize soluble proteins Bound to rER - synthesize secreted or membrane bound proteins - tagged with signal peptide at amino end

Post-translation The new polypeptide is now floating loose in the cytoplasm if translated by a free ribosome. Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. Often translation is not sufficient to make a functional protein, polypeptide chains are modified after translation Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.

Post-translational modifications Trimming Covalent modifications Phosphorylation Glycosylation Hydroxylation Other covalent modifications Formation of disulfide links Addition of carboxyl, acetyl group etc. Addition of prosthetic group

Disulfide bonds form between cysteines Disulfide isomerase works in the ER. In the cytosol most Cystines are in the reduced state partly because of active oxygen radical scavengers. In the ER disulfide bond to another cystine within the target protein.