Gene Expression and Translation

From RNA to Protein: Translation

Overview

• Translation is the process of making protein from RNA.
• It is the second phase of gene expression.
• Messenger RNA (mRNA) information is converted into a protein sequence.
• Proteins give cells their particular function or phenotype.
• The function of cells at a cellular level dictates how the whole organism functions.

The Genetic Code

• The genetic code encodes information for protein synthesis, fundamentally residing in the DNA sequence.
• The DNA sequence is split into three nucleotide chunks called codons.
• One codon encodes for one amino acid in the protein.
• There are four bases, implying 4^3 = 64 possible different sequences of DNA.

Codons

• One start codon: AUG encodes methionine.
• Three termination codons: these signal the end of translation.
• 60 codons for 20 amino acids.
• The genetic code is redundant: most amino acids have more than one codon that can encode them.

Universality and Variation

• The genetic code is almost universal for most forms of life, although there are some variations in codon usage.
• Most amino acids have four codon options, some have a couple, and the start codon has only one.
• Having more options allows for mutations in the DNA sequence that don't impact the amino acid encoded.

The Translation Process

• In prokaryotic cells: translation begins before transcription is finished.
• In eukaryotes: transcription occurs in the nucleus; translation occurs in the cytoplasm.
• Eukaryotic compartmentalization allows for differential regulation.
• Prokaryotes lack a nucleus, so transcription and translation happen in the same space.

Requirements for Translation

• Template RNA.
• Enzymes to activate the process.
• Transfer RNA (tRNA).
• Ribosome.

Transfer RNA (tRNA)

• tRNA acts as an adapter, linking the codon in the mRNA sequence to the amino acid.
• The three nucleotides of complementary RNA in the tRNA is called an anticodon.
• tRNAs recognize codons and bind to specific amino acids, acting as the intermediary.
• Aminoacyl tRNA synthetases attach the specific amino acid to the correct tRNA, creating a charged tRNA.

tRNA Structure

• tRNAs have a similar structure and the base paired regions twist like a DNA molecule.
• At the bottom is the RNA sequence that is complementary to the messenger RNA codon (anticodon).
• One end attaches to a specific amino acid.

Aminoacyl tRNA Synthetases

• Produces charged tRNA in a cycle that requires ATP.
• For every tRNA and amino acid combination, there is a specific aminoacyl tRNA synthetase enzyme.
• This ensures that the correct tRNA is bound to the correct amino acid.
• Example:
  • Alanine binds to the active site of the enzyme.
  • Alanine-specific tRNA binds, a reaction occurs, and alanine is attached to the tRNA molecule.
  • The charged tRNA floats off to take part in translation and the process continues with another alanine and ATP.

The Ribosome

• Messenger RNA is brought into the active site of the ribosome.
• The ribosome has two subunits: small and large, that clamp around the mRNA molecule.
• Once they do that, there are three active sites that are formed: A, P, and E.

Active Sites

• A (aminoacyl tRNA active site): recognizes the charged tRNA.
• P (peptidyl tRNA site): where the reaction occurs, and amino acids are joined.
• E (exit site): where the tRNA exits.

Ribosome Structure

• Ribosome looks like a hand grabbing around the RNA molecule.
• The active sites are adjacent to each other.
• Part of the structure of the ribosome is RNA itself called ribosomal RNA.
• Removing the ribosomal RNA prevents it from working.
• Hypothesis that life actually developed from an RNA world initially before proteins came along.

Translation Process: Initiation, Elongation, and Termination

Initiation

• A complex of proteins/enzymes comes in at the start codon (AUG) with methionine (fMet in prokaryotes) attached to tRNA.
• Small ribosomal subunit is bound to the mRNA and begins the process.
• The start sequence is called the Shine-Dalgarno sequence (anti-SD).
• Positions the small subunit so that the first codon is the first thing it sees.
• The large subunit comes in and the whole thing is set up as a complex around that specific start codon.

Elongation

• Ribosome moves along the RNA in a five to three prime direction.
• Polypeptides grow and are generated within the ribosome and then spill out of the active site once they get long enough.
• Polypeptide is just all the amino acids joined together and are polymerized together with an N and C terminus.

N and C Termini

• N and C termini refer to the amino group (amine) and carboxylic acid group on either end of the amino acid chain.
• Charged tRNAs bring in amino acids one at a time to be stitched onto the growing polypeptide chain.
• Specificity comes from the anticodon and codon base pairing and the aminoacyl tRNA synthetase enzymes.
• A release factor binds at the stop codon acting as a roadblock.
• The ribosome works its way along the RNA and gets to the point where there's a stop codon, and the release factor blocks the whole process.
• The whole complex falls apart, and the small and large subunits are released from the RNA, and the polypeptide is released.
• The peptide chain is then folded up into a fully functioning protein.

Efficiency: Polyribosomes or Polysomes

• Multiple ribosomes run the whole length of the RNA molecule.
• Creates almost like a traffic jam of ribosomes.
• Ribosomes cover an RNA molecule.
• Allows regulation from the RNA production (transcription) and allows the making of lots of copies of a protein from a single RNA molecule.
• Stability of the RNA is dictated by its function.
• Developmental processes may have unstable RNA, while other processes have stable RNA.
• Translation occurs at 12 amino acids a second.