Protein Synthesis

• The document concerns itself with part 2 of "Biotechnology" (presumably a course or textbook).
• Part 2 covers protein, protein synthesis, and protein technology, specifically:
• Composition and structure of proteins
• Protein synthesis
• Analysis of proteins
• Purification of proteins
• The document then pivots to part 2, covering gene regulation:
• Gene regulation in bacteria
• Gene regulation in eukaryotes
• Genetic technology for identifying functional domains in DNA

Protein Synthesis

• The genetic code is universal.
• Codon-anticodon interaction plays a role.
• The ribosome functions as the protein factory.
• Protein synthesis occurs in prokaryotes (like E. coli).
• Protein synthesis also happens in eukaryotes.

Central Dogma of Molecular Biology

• DNA replicates and remains DNA.
• DNA transcribes to RNA.
• RNA translates to proteins.
• Transcriptional activity is influenced by promoter activity.
• RNA transcripts undergo maturation.
• Transcript stability affects polypeptide yield.

Genetic Code

• The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells.
• The genetic code features 64 possible codons, but only 20 amino acids.
• There is a degeneracy code.
• Polymerization of L-amino acids occurs from N-terminus to C-terminus.
• It is a universal code but has some exceptions.
• Codons are distinct from anticodons.

Codon Organization

• The code is organized to minimize the effects of mutations.
• A mutation in the first position might result in an equivalent amino acid.
• Pyrimidine in the 2nd position (HFB).
• Purine in the 2nd position (HFL).
• Third position mutations lead to degeneration.

Codon–Anticodon Interactions

• Inosine can result from deamination.
• Wobble rules apply to base pairing between codons and anticodons:
• G in the anticodon pairs with U or C in the codon
• C in the anticodon pairs with G in the codon
• A in the anticodon pairs with U in the codon
• U in the anticodon pairs with A or G in the codon
• I in the anticodon pairs with A, U, or C in the codon
• These rules allow for some flexibility in base pairing.
• This results in freedom to base pair with the 3' end of the codon.
• The wobble applies only to that position.
• Codon preferences vary per organism.
• Codon usage is not random.
• Start codons include Met-tRNAFMet.
• Prokaryotes: AUG (GUG) (UUG, CUG)
• Eukaryotes: AUG
• Stop codons are:
• UAA.
• UAG.
• UGA.
• They utilize releasing factors.

Ribosomes

• Ribosomes facilitate protein synthesis.
• In prokaryotes the ribosome is 70S, consisting of:
• 23S and 5S rRNAs (34 proteins)
• 16S rRNA (21 proteins)
• In Eukaryotes the ribosome is 80S, consisting of:
• 28S, 5.8S, and 5S rRNAs (~45 proteins)
• 18S rRNA (~30 proteins)
• Ribosomes contain the A (aminoacyl) site, P (peptidyl) site, and E (exit) sites.

Protein Synthesis Steps in Prokaryotes

• Initiation begins with N-formyl-methionine.
• The formyl group is installed after transfer of AZ to the tRNA adaptor.
• There are two different tRNAMet, one of which permits formylation.
• Both tRNA recognize AUG.
• tRNAFMet also recognizes AUG.
• The initiation codon is therefore AUG.
• After the start of protein synthesis, methionine is deformylated.
• Deformylase is responsible.
• An initiation complex forms containing:
• the ribosomal 30S subunit
• the mRNA
• the initiating fMet-tRNAFMet
• After the initiation complex forms, the 50S subunit binds.
• This leads to a complete 70S ribosome.
• An efficient initiation requires a ribosome-binding-site (RBS) on the mRNA.
• mRNA has a Shine-Dalgarno (SD) sequence, followed by an AUG start codon.
• This is located 8–13 nucleotides downstream.
• Bacteria are polycistronic; one SD for each start codon.
• SD-sequence is recognized by 3’-end of ribosomal 16S rRNA (in 30S subunit).
• This positions the 30S subunit correctly on the mRNA.
• Two RNA-RNA interactions facilitate the binding:
• mRNA and 16S rRNA, at the SD sequence
• tRNAFMet/fMet and the initiation codon AUG
• Initiation factors are also required.
• Binding of the 50S subunit requires a GTP molecule.
• IF1 blocks the ribosomal A-site.
• IF1 makes the binding of IF2 and IF3 easier.
• IF2 promotes the binding of the initiator tRNA.
• IF3 prevents the association of the 50S subunit with the 30S subunit.
• IF3 promotes 30S mRNA binding.
• The initiating tRNA binds to the P-site.

Elongation

• mRNA molecules runs through ribosomes in the 5'-3' direction.
• Each ribosome has three tRNA-binding spots:
• Peptidyl (P) site
• Aminoacyl (A) site
• Exit (E) site
• Elongation requires 2 GTP molecules per incorporated amino acid.
• Binding an amino acid-tRNA complex requires EF-Tu and EF-Ts.
• Translocation from A site to P site requires EF-G.
• The growing polypeptide chain immediately adopts secondary/tertiary structures.
• Several ribosomes can translate an mRNA at the same time.

Termination in Prokaryotes

• Stop codons inlcude UAA, UAG, or UGA.
• Requires releasing factors (RF).
• RF1
• RF2
• RF3 stimulates RF1 and RF2 activity, consuming 1 GTP.

Exported Proteins

• Proteins that migrate across the cell membrane have a signal peptide.
• Properties of a signal peptide:
• 15-30 amino acids long (extra)
• Located at the NH2-end
• Includes one or more "basic" amino acids
• Includes interior hydrophobic amino acids
• Directs a protein across the membrane.
• Migration across the cell membrane occurs either co-translationally or post-translationally.
• Signal peptides and sequences determine the export fully.
• Signal peptides can form antiparallel hairpends.
• Export utilizes SRP-dependent pathways and the SRP receptor in the cell membrane (FtsY).

Explore the role of ribosomes, codon degeneracy, and the central dogma in protein synthesis. Learn about start codons, translation initiation in bacteria, and the impact of mutations in the Shine-Dalgarno sequence. Also, understand the Shine-Dalgarno sequences in bacterial mRNA.