BIOL 2416 CH 6: Translation

What is a protein? A protein consists of 1 or more polypeptides A polypeptide is a polymer of amino acids bound together by peptide bonds The amino acid sequence dictates the 3-D shape of the protein (folding instructions) The 3-D shape of a protein determines its function

A functional protein consists of one or more polypeptides that have been precisely twisted, folded, and coiled into a unique shape. It is the order of amino acids that helps to determine what the three-dimensional conformation will be.

Translation = protein synthesis = forging peptide bonds between amino acids Done by ribosomes in cytosol (eukaryotes) Ribosomes hook amino acids together by covalent peptide bonds according to mRNA sequence tRNAs deliver correct amino acids to ribosomes 20 amino acids N-terminus = beginning of polypeptide (NH2 / NH3 + ) C-terminus = end (COOH / COO - )

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 6.1 General structural formula for an amino acid

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Sinceamino acids differ only by their side chains (R groups), it is the side chain that gives each amino acid its unique chemical properties.

Polymers (polypeptides) are formed from monomers (amino acids) by dehydration reactions catalyzed by ribosomes.

The primary structure of a protein is its unique sequence of amino acids. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

The secondary structure of a protein results from hydrogen bonds at regular intervals along the polypeptide backbone. –Typical shapes that develop from secondary structure are coils (an alpha helix) or folds (beta pleated sheets). Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Fig. 5.20

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings Tertiary structure is determined by a variety of interactions among R groups and between R groups and the polypeptide backbone.

Quarternary structure results from the aggregation of two or more (identical or different) polypeptide subunits. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

The triplet genetic code 1 codon = 3 adjacent mRNA nucleotides (sense, start and stop codons) No skipping bases No overlap Almost universal Unambiguous (means what it says) Degenerate: 3-rd base pair wobble (more than one way to call for same amino acid) 3 possible reading frames:

3 possible reading frames for the same sequence: WHYDIDTHEREDCATEATTHEFATRAT ? Prokaryotes: use a Ribosome Binding Site (Shine-Dalgarno sequence) to find the first AUG Eukaryotes: use a scanning (sliding) model to find first AUG; first AUG embedded in diagnostic Kozak sequence

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig. 6.8 The genetic code

A Translation Example mRNA: 5’ AUGACUAGGUCAUUUUAG 3’ release factor Protein: N- Met-Thr-Arg-Ser-Phe -C

A tRNA molecule consists of a strand of about 80 nucleotides that folds back on itself to form a three-dimensional structure. –It includes a loop containing the anticodon and an attachment site at the 3’ end for an amino acid. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

tRNAs interact with mRNA codons by antiparrallel, complementary tRNA anticodon-mRNA codon binding Wobble hypothesis explains degeneracy (redundancy) of code

Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase. The 20 different synthetases match the 20 different amino acids. –Each has active sites for only a specific tRNA and amino acid combination. –The synthetase catalyzes a covalent bond between them, forming aminoacyl- tRNA.

Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules. –The P site holds the tRNA carrying the growing polypeptide chain. –The A site carries the tRNA with the next amino acid (#2 and up). –Empty tRNAs leave the ribosome when at the E site. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Translation can be divided into three stages: 1. initiation 2. elongation a. codon recognition b. peptide bond formation c. translocation 3. termination All three phase require protein “factors” that aid in the translation process (initiation, elongation, release factors) Both initiation and chain elongation (codon recognition and translocation) require energy provided by the hydrolysis of GTP. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Prokaryotic Translation Initiation: 1.30S ribosomal subunit + mRNA + initiator fMet-tRNA + initiation factors get together to form a 30S initiation complex 2.50S ribosomal subunit joins the 30S initiation complex, releasing the initiation factors. GTP is turned into GDP + Pi, releasing the necessary energy to make this happen 3.fMet-tRNA sits in the P site. We are ready to grow the polypeptide according to the mRNA codon instructions. Can bind 16S rRNA part of 30S Different in eukaryotes: 40S+60S=80S ribosomes, met-tRNA, scanning model Instead of RBS/Shine-Dalgarno mechanism

The three steps of elongation continue codon by codon to add amino acids until the polypeptide chain is completed. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings (ribosome hopping over by 1 codon) (between last and next-to-last amino acid) (tRNA delivers next amino acid in A site; tRNA anticodon binds mRNA codon) (empty tRNA leaves E site; Growing polypeptide now in P site) needs GTP energy (EF-Tu-GDP) (EF-G-GDP)

Termination occurs when one of the three stop codons (UGA,UAA,UAG) reaches the A site. A release factor (RF1) binds to the stop codon in the A site and cuts off the polypeptide. Binding of RRF Ribosome Recycling Factor recruits EF-G-GDP RRF releases now-empty tRNA EF-G-GDP releases RRF Ribosomal subunits and mRNA go separate ways All can be re-used Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Prokaryotes can transcribe and translate the same gene simultaneously (txn and tln are “coupled” to form polysomes): In eukaryotes, transcription is separated by the nuclear envelope.

In eukaryotes, free ribosomes may end up bound to rough ER (when making membrane/secretory proteins): Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings