9. The Molecular Biology of Translation

9. The Molecular Biology of Translation

9.1 Polypeptides Are Composed of Amino Acid Chains That Are Assembled at Ribosomes

Translation is the process by which ribosomes synthesize polypeptides using messenger RNA (mRNA) as a template. Polypeptides are linear chains of amino acids, and their assembly is fundamental to gene expression.

• Twenty different amino acids serve as the building blocks of polypeptides.

• Covalent peptide bonds form between amino acids to create a polypeptide chain.

• The distinctive features of amino acids (side chains or R-groups) determine their chemical reactivity and whether they are hydrophobic or hydrophilic.

Amino Acid Structure

All amino acids share a common structure but differ in their side chains, which confer unique properties.

• Each amino acid has a central (alpha) carbon, an amino group (-NH2), and a carboxyl group (-COOH).

• Peptide bond formation is catalyzed by the ribosome, linking the carboxyl group of one amino acid to the amino group of the next, releasing water (a condensation reaction): $\text{Amino acid}_1 + \text{Amino acid}_2 \rightarrow \text{Dipeptide} + H_2O$

• The R-group (side chain) is unique for each amino acid and determines its properties (e.g., charge, polarity).

Table: Amino Acids Grouped by Their Side Chain Properties

Nonpolar Side Chains	Polar Side Chains	Electrically Charged Side Chains
Alanine (Ala, A) Cysteine (Cys, C) Glycine (Gly, G) Isoleucine (Ile, I) Leucine (Leu, L) Methionine (Met, M) Phenylalanine (Phe, F) Proline (Pro, P) Tryptophan (Trp, W) Valine (Val, V)	Asparagine (Asn, N) Glutamine (Gln, Q) Serine (Ser, S) Threonine (Thr, T) Tyrosine (Tyr, Y)	Acidic: Aspartic acid (Asp, D), Glutamic acid (Glu, E) Basic: Arginine (Arg, R), Histidine (His, H), Lysine (Lys, K)

Polypeptide and Transcript Structure

Polypeptides are synthesized by ribosomes, which read mRNA sequences and assemble amino acids in the correct order.

• Ribosomes are ribonucleoprotein complexes containing ribosomal RNAs (rRNAs) and proteins.

• Translation proceeds in the 5'-to-3' direction along the mRNA, reading each triplet codon.

Messenger RNA (mRNA)

The sequence of mRNA determines the amino acid sequence of the resulting polypeptide.

• Translation boundaries are defined by a start codon (N-terminus) and a stop codon (C-terminus).

• The 5' untranslated region (5' UTR) and 3' UTR are segments of mRNA outside the coding region.

Polypeptide Structure

Polypeptides exhibit four levels of structural organization, each contributing to the final protein's function.

• Primary structure: The linear sequence of amino acids in the polypeptide chain.

• Secondary structure: Local folding patterns stabilized by hydrogen bonds, such as alpha helices and beta-pleated sheets.

• Tertiary structure: The overall three-dimensional shape formed by interactions among R-groups; dependent on primary and secondary structure.

• Quaternary structure: The association of two or more polypeptide chains into a functional protein complex.

Table: Polypeptide Structure

Level	Description	Stabilized By	Example (Hemoglobin)
Primary	Sequence of amino acids	Peptide bonds	One alpha helix
Secondary	Formation of helices and sheets	Hydrogen bonding	One of hemoglobin's subunits
Tertiary	Three-dimensional folding	R-group interactions, disulfide bonds	Hemoglobin subunit
Quaternary	Multiple polypeptides	Interactions between subunits	Hemoglobin (4 subunits)

Ribosome Structures

Ribosomes are the molecular machines responsible for protein synthesis in all living cells.

• They bind mRNA and identify the start codon.

• They facilitate base pairing between mRNA codons and tRNA anticodons.

• They catalyze peptide bond formation between amino acids.

Ribosome Composition

Ribosomes are composed of two subunits, each containing rRNA and proteins. Their composition and size differ among domains of life.

• Large and small subunits are measured in Svedberg units (S), reflecting their size and shape.

• Bacterial ribosomes: 70S (30S small + 50S large)

• Eukaryotic ribosomes: 80S (40S small + 60S large)

Ribosomes of E. coli

• 30S small subunit: 21 proteins, 16S rRNA

• 50S large subunit: 32 proteins, 5S and 23S rRNAs

• Fully assembled ribosome: 70S

Important Regions of Ribosomes

• P site (peptidyl site): Holds the tRNA with the growing polypeptide chain.

• A site (aminoacyl site): Binds incoming tRNA carrying the next amino acid.

• E site (exit site): Where tRNA exits after delivering its amino acid.

• The large subunit contains a channel for the emerging polypeptide.

Eukaryotic Ribosomes

• 40S small subunit: ~34 proteins, 18S rRNA

• 60S large subunit: 49 proteins, 5S, 5.8S, and 28S rRNAs

• Fully assembled ribosome: 80S

The Three-Dimensional Structure of the Ribosome

Advanced imaging techniques, such as cryo-electron microscopy, are used to study ribosome structure.

• Ribosomes are ~25 nm in diameter.

• Cryo-EM preserves native structure and allows 3D reconstruction.

9.2 Translation Occurs in Three Phases

Translation is divided into three main phases: initiation, elongation, and termination. While the overall process is conserved, there are differences between bacteria and eukaryotes, especially in initiation.

• Initiation: Assembly of the translation machinery at the start codon.

• Elongation: Sequential addition of amino acids.

• Termination: Release of the completed polypeptide at a stop codon.

Translational Initiation

• The small ribosomal subunit binds near the 5' end of mRNA and locates the start codon.

• The initiator tRNA binds to the start codon.

• The large subunit joins to form the complete ribosome.

Initiation Factors and Energy

• Initiation factor proteins regulate ribosome assembly and initiator tRNA binding.

• GTP provides energy for initiation.

• tRNAs carrying amino acids are charged tRNAs; those without are uncharged.

Bacterial Translational Initiation

• Six components: mRNA, small and large ribosomal subunits, initiator tRNA, three initiation factors (IF1, IF2, IF3), and GTP.

• IF3 prevents premature association of subunits.

• The small subunit-IF3 complex scans for the start codon.

The Shine-Dalgarno Sequence

• The preinitiation complex forms when the 16S rRNA pairs with the Shine-Dalgarno sequence on mRNA.

• The Shine-Dalgarno sequence is a purine-rich region upstream of the start codon.

• It aligns the ribosome for correct translation initiation.

The Second Step of Initiation

• The initiator tRNA (carrying N-formylmethionine, fMet) binds the start codon at the future P site.

• IF2 and GTP facilitate this binding; IF1 joins to form the 30S initiation complex.

The Final Step of Initiation

• The 50S subunit joins the 30S subunit, driven by GTP hydrolysis.

• IF1, IF2, and IF3 dissociate, forming the 70S initiation complex.

Eukaryotic Translational Initiation

• The 40S subunit associates with eukaryotic initiation factors (eIF1, eIF1A, eIF3) to form the preinitiation complex.

• The preinitiation complex joins with initiator tRNA and eIF5.

Later Steps of Eukaryotic Initiation

• mRNA joins the preinitiation complex to form the initiation complex.

• The complex scans the 5' UTR for the start codon (usually the first AUG).

• The Kozak sequence (5'-ACCAUGG-3') helps identify the correct start codon.

• Recruitment of the 60S subunit and GTP hydrolysis complete initiation.