Genetic Code and Translation

• The central dogma involves DNA undergoing transcription to produce RNA, which then undergoes translation to produce protein.

Peptide Bond Formation

• A peptide bond forms when the carboxyl group of one amino acid reacts with the amino group of another, releasing water (H₂O).
• The resulting bond links the amino acids together.

Amino Acids and Polypeptide Chains

• Amino acids are linked together by peptidyl bonds
• Polypeptide chains exhibit polarity: one end has a free amino group (N-terminus), and the other has a free carboxyl group (C-terminus).
• Convention dictates that amino acid sequences are written from the N-terminus to the C-terminus.

Protein Structure

• Primary structure describes the sequence of amino acids held together by peptide bonds in the polypeptide chain.
• Secondary structure arises from hydrogen bonds between atoms of the polypeptide backbone, forming structures like α-helices and β-sheets.
  • α-helices: Hydrogen bonds form between every fourth amino acid.
  • β-sheets: Hydrogen bonds form between amino acids in parallel strands.
• Tertiary structure is the overall 3D shape of a protein, resulting from interactions between amino acids within a polypeptide chain.
  • Bonds in tertiary structure include hydrogen bonds, ionic bonds, hydrophobic interactions, and disulfide bonds between cysteine residues.
• Quaternary structure involves interactions between amino acids from different polypeptide chains.

RNA Types and Their Roles

• Messenger RNA (mRNA) carries genetic information for protein synthesis.
• Ribosomal RNA (rRNA) is a component of ribosomes.
• Transfer RNA (tRNA) is involved in transferring amino acids to the ribosome.
• Micro RNA (miRNA) has a role in gene regulation.

Deciphering the Genetic Code

• Nirenberg, Leder, Mathaei, and Khorana deciphered throughout the 1960's,
• 3 nucleotides make up a codon.
• There are 20 different amino acids
• 64 possible codons exist (4³=64).
• Genes utilize a non-overlapping genetic code, so a base mutation changes only one codon.

Characteristics of the Genetic Code:

• Made up of trinucleotides, codons
• Consists of trinucleotide codons.
• 64 possible codons specify only 20 amino acids.
• includes 3 stop codons and 61 codons for 20 amino acids
• The code is redundant with multiple codons for the same amino acid.
• The code is non-overlapping.
• The code is non-punctuated.
• The code is universal.

Experiments and Codon Determination

• UUU codes for phenylalanine.
• GUC codes for valine
• Poly-U mRNA codes for polyphenylalanine.
• Synthetic mRNAs are used to produce polypeptides with varying sequences.
  • Repeating dinucleotides produce polypeptides with two alternating amino acids.
  • Repeating trinucleotides yield three polypeptides, each with a single amino acid type.
  • Repeating tetranucleotides may produce polypeptides with four repeating amino acids.

Identifying Open Reading Frames (ORFs)

• Identify the coding strand.
• The sequence of amino acids is given from the amino to the carboxyl terminal end from left to right by convention.
• Start codons (typically ATG) indicate the beginning of an ORF.
• An ORF is a series of codons coding for amino acids without any stop codons.

Eukaryotic vs. Prokaryotic Translation

• Eukaryotic mRNA is monocistronic
  • Eukaryotic mRNA contains a 5'-untranslated region (5'-UTR) and a 3'-untranslated region (3'-UTR)
• Prokaryotic genes are organized into operons.
  • operons encode proteins with related functions and share a promoter.
• Prokaryotic mRNA is polycistronic

Transfer RNA (tRNA)

• tRNA + amino acid = amino acyl tRNA.
• Amino acyl tRNA synthetase specifically attaches an amino acid to its corresponding tRNA
• The 3' base of a codon in the mRNA interacts with with the 5' base of the anticodon in the tRNA
• One tRNA with a given anticodon interacts with several different codons.

Wobble Pairing

• Flexibility exists in the interaction which occurs between the 3' base of a codon in the mRNA and the 5' base of the anticodon.

tRNAs and Codon Interactions

• Some tRNAs interact with only one codon, while others interact with several.
• The cell contains fewer than 61 tRNA types but more than 20.

Ribosome Structure

• Bacterial (E. coli) ribosomes:
  • Are composed of a large (50S) and small (30S) subunit. -large subunit contains 23S rRNA (2904 nucleotides) and 5S rRNA (120 nucleotides) and 32 proteins. -The small subunit contains 16S rRNA (1541 nucleotides) and 21 proteins.
• Eukaryotic (mammalian) ribosomes:
  • Are composed of a large (60S) and small (40S) subunit.
    • large subunit containing 28S rRNA (4718 nucleotides), 5.8S rRNA (160 nucleotides) and 5S rRNA (120 nucleotides) and 50 proteins.
  • small subunit contains 18S rRNA (1874 nucleotides) and ≈35 proteins.
• Ribosomes sizes are measured in Svedberg units (S), which indicate sedimentation rate.

Ribosome Binding Sites

• A-site :
  • Amino acyl tRNA initially binds to the ribosome.
  • Site interacts with the corresponding codon in mRNA - (except for the Met-tRNA binding to the start codon).
• P-site :
  • Site for peptidyl-tRNA is located where the new amino acyl-tRNA binds to the A site.
• E-site : -Exit site where empty tRNA leaves the ribosome.
• Amino acyl tRNA: tRNA with an amino acid attached to the 3' end is also called charged tRNA.
• Peptidyl-tRNA: tRNA with a peptide attached to its 3' end.
• Empty tRNA: tRNA without an amino acid or peptide bound.

Translation Steps

• In bacteria, the initiator tRNA carries a formyl-Met, whereas in eukaryotes, initiator tRNA carries a Met.
• The mRNA binds to the small ribosomal subunit. mRNA is positioned by the start codon in the P site.
  • In prokaryotes, a conserved sequence within the 5'-UTR of the mRNA (Shine-Dalgarno) binds to a rRNA within the small ribosomal subunit.
  • In Eukaryotes, the cap at the 5' end of the mRNA allows in to bind to the small ribosomal subunit. initiator binds with the start codon at the P.
• The large ribosomal subunit then assembles, consuming energy from GTP hydrolysis.
• Initiation factors facilitate this process and differ in prokaryotic versus eukaryotic cells.

Elongation in Detail:

• A new amino acyl-tRNA enters, binding the next codon at the A site.
• Amino acid/peptide on the P-site tRNA is transferred to the charged tRNA at the A site.
• A peptide forms, resulting in peptidyl tRNA at the A site and empty tRNA at the P site.
• The ribosome undergoes translocation along mRNA to its 3' end.

Translocation:

• The Peptidyl-tRNA on the A-site moves to the P-site.
• Empty tRNA on the P-site moves to the E-site.
• The cycle repeats with each addition of amino acids.
• GTP hydrolysis provides elongation energy.

Termination

• When a stop codon reaches the A site, no tRNAs can bind to it, causing translation to stop.
• The completed polypeptide is released from the tRNA at the P site.
• The mRNA detaches from the ribosome.
• The ribosomal subunits separate.
• Energy is used during Termination
• Termination process needs release factors to allow dissociation.

Polysomes

• Translation generally involves multiple ribosomes binding to a single mRNA, thus creating polyribosomes or polysomes.
• In bacteria, transcription and translation occur simultaneously, polyribosomes are seen on mRNA simultaneously prior to translation completion.