Translation�

• mRNA 🡪 protein
• Process of mRNA converting to a protein
• Occurs in the cytoplasm – ribosome

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The DNA stores information about protein synthesis, and the body requires the synthesis of the right proteins in accordance with its needs. These proteins are produced by DNA through RNA and are referred to as the Central Dogma.

tRNA

• Translator of mRNA’s message is tRNA – transfer RNA
• 80 nucleotides long
• Hairpin shape – L shaped
• One end contains an anticodon which pairs with the codon on the mRNA
• Codons determine which amino acid is coded for by the DNA
• The other end contains an amino acid attachment site
• Aminoacyl-tRNA synthetase attaches the correct amino acid to the tRNA

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tRNA

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Why we need t-RNA in Translation

• The DNA is transcribed into mRNA on the basis of their complementarity. Translation is the process of conversion of nucleic acid information into amino acids. There is no complementarity between amino acids and mRNA. Hence, translation is not controlled by complementarity but by the genetic code. Since amino acids cannot read this genetic code, they are dependent on an adapter molecule. This adapter molecule is called tRNA (transfer RNA).

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• In translation, the codons of an mRNA are read in order (from the 5' end to the 3' end) by molecules called transfer RNAs, or tRNAs.

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• Each tRNA has an anticodon, a set of three nucleotides that binds to a matching mRNA codon through base pairing. The other end of the tRNA carries the amino acid that's specified by the codon.

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Ribosomes/ rRNA

• Pair codons on mRNA with anticodons on tRNA to form polypeptides
• Made of large and small subunits
• rRNA – ribosomal RNA
• Made in the nucleolus
• Contain multiple binding sites
• mRNA binding site
• P site – peptidyl – tRNA site
• A site – aminoacyl – tRNA site
• E site – exit site

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Within the ribosome, the rRNA molecules direct the catalytic steps of protein synthesis — the stitching together of amino acids to make a protein molecule. In fact, rRNA is sometimes called a ribozyme or catalytic RNA to reflect this function.

Ribosomes

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Making a protein - Summary

• Initiation
• Small subunit binds to mRNA
• Start codon AUG – methionine at P site
• Elongation
• A site recognizes codon and pairs with correct tRNA
• Peptide bond forms between the carboxyl end of the polypeptide at the P site and amino acid at the A site
• Amino acid in the A site translocates to the P site
• Termination
• Stop codon is reached at the A site
• UAA, UAG, UGA
• Release factors free the polypeptide from the ribosome

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Making a Protein/ Translation�

Intitiation

• The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IF-1, IF-2, IF-3) that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAf^Met).
• The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli.
• In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome.
• This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

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Elongation

• In prokaryotes and eukaryotes, the basics of elongation of translation are the same.
• In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites:
• The A (aminoacyl) site binds incoming charged aminoacyl tRNAs.
• The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA.
• The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids.

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Elongation continues

• Elongation proceeds with single-codon movements of the ribosome each called a translocation event.
• During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal.
• Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3′ direction.
• Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA.
• The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit.

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Elongation continues

• As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled.
• Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors.
• Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

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Translation

• The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA.
• On aligning with the A site, these nonsense codons are recognized by release factors (RF1,RF2,RF3) in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide.
• The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex.
• In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes.

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Prokaryotes vs. Eukaryotes

Prokaryotes

• No nucleus
• Tanscription and translation same location
• Smaller ribosomes

Eukaryotes

• Locations:
• Free cytosol Ribosomes: Stay in the cell somewhere (free floating, mitochondria, etc)
• Rough ER Ribosomes: SRP sends ribosome to go to ER, becomes protein in endomembrane system, or cell membrane protein or is secreted from cell (all in vesicles)
• Nucleus
• Pre-mRNA 🡪 mRNA
• transcription
• Larger ribosomes
• translation

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Translation

Information transmission

• 4 bases in DNA/RNA to 20 amino acids in proteins
• “translation” since the chemical language is different
• How many nucleotides needed to specify each amino acid?
• Three basepairs of DNA = 64 combinations

Processed (e.g., spliced) mRNA is read in groups of three nucleotides

• called codons:
• Codon Explanation: A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis.

redundancy of codons for different amino acids à typically the last nucleotide is variable

• Anticodon Explanation: An anticodon is a trinucleotide sequence complementary to that of a corresponding codon in a messenger RNA (mRNA) sequence. An anticodon is found at one end of a transfer RNA (tRNA) molecule.
• three possible reading frames depending on starting nucleotide

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transfer RNAs (tRNAs) are the intermediates between nucleotides and amino acids

• about 80 nucleotides long
• have specific 3D shape, like an “L”
• at one end: anticodon that base-pairs with mRNA
• at the other end: covalently coupled amino acid
• different tRNAs for each amino acid type
• base pairing weakest at third “wobble” nucleotide (why it is most variable)
• tRNAs are charged with an amino acid by aminoacyl tRNA synthetases that ensure correct addition of individual amino acids to corresponding tRNA

The synthesis of proteins is choreographed by large molecular machines called ribosomes

• large and small subunits = ~82 proteins (1/3) plus 4 ribosomal RNA (rRNA) strands (2/3)
• overall structure and catalytic activity dictated by RNA 🡪 ribozyme