Study Notes on Translation and Protein Synthesis

Translation Notes

RNA and Protein Synthesis

  • RNA (Ribonucleic Acid) is critical for making proteins.

Objectives of Translation Study

  • Understand Requirements for Protein Synthesis
    • Ribosome
    • mRNA (messenger RNA)
    • Soluble Protein Factors
    • Aminoacyl-tRNA
  • Understand the Genetic Code
    • Role of tRNA in Protein Synthesis
  • Charging of Amino Acids to Cognate tRNAs
    • Role of Aminoacyl-tRNA Synthetases
  • Ribosome Function
    • Engine for Protein Synthesis
    • Peptidyl Transferase Reaction
  • Stages of Protein Synthesis
    • Initiation
    • Elongation
    • Termination
  • Compare Protein Synthesis in Prokaryotes and Eukaryotes
    • Differences explored by the use of antibiotics

Translation of RNA into Proteins

  • The problem is how a linear nucleotide sequence in RNA (mRNA) is converted into a linear amino acid sequence in proteins.
  • Example mRNA sequence:
    • 5’-CAGACCAUGAGUGGAU… (n)nUGA… (n)-3’
  • Resulting Protein Sequence: MET-SER-GLY-CYS-AA5-AAn
  • The process called “Translation” is defined by the “Genetic Code.”
    • Translation serves as the framework for the Central Dogma that states RNA specifies Protein.

Requirements for Protein Synthesis

  1. Ribosome
  2. Aminoacyl-tRNAs (charged tRNAs)
  3. mRNA
  4. Soluble protein factors including:
    • IF-1 (Initiation Factor 1)
    • IF-2 (Initiation Factor 2, a GTPase)
    • IF-3 (Initiation Factor 3)
    • EF-Tu (Elongation Factor Tu, a GTPase)
    • EF-Ts (Elongation Factor Ts)
    • EF-G (Elongation Factor G, a GTPase)
    • RF-1 (Release Factor 1)
    • RF-2 (Release Factor 2)
    • RF-3 (Release Factor 3, a GTPase)
    • RRF (Ribosome Recycling Factor, a GTPase)

Translation Mechanism and GTPases

  • In E. coli:
    • GTP is hydrolyzed to provide energy for translation:
    • E•GTP → E’•GDP + H2O + Pi
  • GTPase functions:
    • GAP (GTPase-Activating Protein)
    • GRF (GTPase-Releasing Factor)
  • GTPases are crucial, acting as motors driving the process of protein synthesis forward, coupled with conformational changes.

mRNA Structure and Function

  • m7Gppp- cap at the 5' end
  • AUG start codon and UAA stop codon examples:
  • mRNA sequence structure includes:
    • 5’ UTR (Untranslated Region)
    • ORF (Open Reading Frame)
    • 3’ UTR
  • Characteristics of Eukaryotic mRNA:
    • Typically monocistronic (encodes one polypeptide)
    • Usually spliced
    • Capped at the 5’ end and polyadenylated at the 3’ end
    • The sequence of the protein is framed in an open reading frame (ORF) dictated by the first AUG and terminating at one of three stop codons (UAA, UAG, UGA).

The Genetic Code

  • General Features of the Genetic Code:
    • It is a triplet code, specified by DNA and read from RNA
    • Each triplet (set of three nucleotides) is termed a “codon”
    • With 4 bases (A, U, C, G), there are 64 possible combinations (4^3)
    • 61 coding (sense) codons specify one of the 20 common amino acids
    • 3 non-coding (nonsense) codons specify a stop signal
    • The genetic code is degenerate: multiple codons can code for the same amino acid.

Examples of Codon Reading

  • Codon table for specific amino acids:
    • UUU - Phe (Phenylalanine)
    • UUC - Phe
    • UUA - Leu (Leucine)
    • AUG - Met (Methionine) - Start codon
    • UAA, UAG, UGA - Stop codons

Important Features of the Genetic Code

  • Read as ribonucleotide triplets
  • Degenerate (multiple codons for some amino acids, reducing errors)
  • Universal (sequence gives the same protein product across most organisms)
    • Exceptions include variations in mitochondrial code and species-specific codon usage preferences.

Types of Mutations

  • Mutation: a change in the DNA sequence
  • Types include:
    • Point Mutation: change in a single base pair
    • Silent Mutation: does not affect expressed protein (typically Polymorphisms)
    • Mis-sense Mutation: changes that lead to different amino acids
    • Nonsense Mutation: introduces a stop codon
    • Read-through Mutation: removes a stop codon
    • Insertion/Deletion Mutations: disrupts reading frame
    • Others: in regulatory regions or splicing and chromosomal rearrangements

Translation Process Overview

  • Phases of translation include
    • Initiation
    • Elongation
    • Termination
    • Key Elements: growing polypeptide chain with tRNA interacting with mRNA within ribosome

Ribosome Structure and Function

  • Ribosome in E. coli: 70S (30S small subunit and 50S large subunit)
  • Ribosome in Eukaryotes: 80S (40S small subunit and 60S large subunit)
  • Two primary views of ribosome function:
    • Proteins do the work, RNA serves merely as scaffolding.
    • RNA is the actual catalytic component (ribozyme).

Peptidyl Transferase Reaction

  • The process of forming peptide bonds between amino acids within the ribosome.

Initiation in Prokaryotes

  • Initiation starts with the recognition of the start codon (AUG) by fMet-tRNA^fMet in bacteria, which is different from internal Met sequences.
  • The Shine-Dalgarno sequence guides the ribosome to the AUG initiation site by base-pairing with rRNA.

Elongation Steps

  • 3 Steps in Elongation:
    a) Binding of aminoacyl-tRNA
    b) Formation of peptide bond (transpeptidation)
    c) Translocation (shift by one codon)

Eukaryotic Translation Differences

  • Eukaryotic initiation requires more initiation factors and involves mRNA processing not seen in prokaryotes, with a different initiation signal (AUG) at the beginning of the ORF.

Termination of Translation

  • Termination codons (UAA, UAG, UGA) are recognized by release factors (RF-1, RF-2) which help disassemble the ribosome and release the polypeptide.

Antibiotics and Protein Synthesis Inhibition

  • Antibiotics are valuable for distinguishing between prokaryotic and eukaryotic protein synthesis. Examples include:
    • Streptomycin: causes misreading and inhibits initiation in prokaryotes.
    • Erythromycin: inhibits translocation in prokaryotes.
    • Tetracycline: prevents aminoacyl-tRNA binding in prokaryotes.
    • Chloramphenicol: inhibits peptidyl transferase activity.
    • Puromycin: acts as an analog and inhibits elongation.
    • Cycloheximide: inhibits peptidyl transferase in eukaryotes.

Conclusion

  • In prokaryotes, translation begins immediately after transcription, reflecting the coordinated nature of these processes, with polypeptides synthesized directionally along the mRNA.
  • The electron micrograph may help visualize this process concerning the mRNA’s 5'-3' polarity.