Protein Synthesis - Translation

Protein Synthesis – Translation

• Translation is the process where information encoded in mRNA is translated into a sequence of amino acids, forming a polypeptide chain. This is the final step in gene expression.

• There is colinearity between DNA and proteins, meaning the sequence of nucleotide triplets in DNA corresponds directly to the sequence of amino acids in the protein.

• Example:-

  • Coding strand DNA: 5' A T G A A A T T T C C C 3'

  • Template strand DNA: 3' T A C T T T A A A G G G 5'

  • mRNA (transcription product): 5' A U G A A A U U U C C C 3'

  • Protein (translation product): N-Met-Lys-Phe-Pro-C-end

Translation – Introduction

• Template: mRNA, which carries the genetic code from the DNA.

• Translation apparatus: ribosomes, which are complex molecular machines composed of ribosomal RNA (rRNA) and ribosomal proteins.

  • Prokaryotes have 70S ribosomes, while eukaryotes have 80S ribosomes. The 'S' stands for Svedberg units, a measure of sedimentation rate during centrifugation, reflecting size and shape.

• Rate of translation: 10-20 amino acids per second, with 10-15 ribosomes per mRNA, forming polyribosomes or polysomes, which enhance the efficiency of translation.

• Fidelity of translation: approximately 1 error per 2000 incorporated amino acids. Incorrectly synthesized proteins are usually degraded by proteases.

Localization of Translation

• Cytoplasm: occurs in both prokaryotes and eukaryotes.

  • In eukaryotes, the initiation of translation for all proteins (cytoplasmic, nuclear, mitochondrial, and peroxisomal) occurs in the cytoplasm.

• Rough Endoplasmic Reticulum (RER) and Golgi apparatus: Synthesis of secretory, plasmalemal, and lysosomal proteins continues here. These proteins are translocated into the ER lumen for folding, modification, and transport.

• Mitochondria: utilize 70S ribosomes for the synthesis of mitochondrial proteins (13 in human beings) coded by mtDNA.

• Chloroplasts: also utilize 70S ribosomes, reflecting their evolutionary origin from bacteria.

Genetic Code

• Codon: a triplet of nucleotides in mRNA that codes for one amino acid. There are 64 codons in total.

• Start codon: AUG initiates translation and codes for methionine (Met). It also sets the reading frame for translation.

• Termination codons: UAA, UAG, and UGA signal the termination of translation. They do not code for any amino acid.

  • Some codons are bifunctional; for example, UGA can code for stop or selenocysteine under specific conditions.

• Universal: the genetic code is valid for all organisms, except for mitochondrial genetic code which has some differences, such as different start and stop codons.

• Degenerated: more than one codon can code for a single amino acid (except for Met and Trp, which have only one codon each). This redundancy minimizes the impact of mutations.

• Non-overlapping: one nucleotide is part of only one codon. This ensures that the reading frame is maintained.

Reading Frame

• Open Reading Frame (ORF): Determined by a start codon (AUG) and a stop codon, producing a polypeptide of the expected size. It indicates the protein-coding region of a gene.

• Closed Reading Frame: Also determined by a start codon (AUG) and a stop codon, but produces a short, nonfunctional polypeptide.

• Example:

  • Sequence of mRNA: 5'-UCAUGCUCACUGUCUUG-3'

  • Reading frame 1: UCA UGC UCA CUG UCU UG

  • Reading frame 2: CAU GCU CAC UGU CUU G

  • Reading frame 3: AUG CUC ACU GUC UUG

Genetic Code Table

The table shows the relationship between mRNA codons and amino acids. U, C, A, and G represent the nucleotide bases. Term* denotes termination codons.

Interaction - Codon (mRNA) – Anticodon (tRNA)

• Wobble pairing describes the flexible base pairing between a codon in mRNA and an anticodon in tRNA at the third nucleotide position. This flexibility allows a single tRNA to recognize more than one codon.

Mutations – Impact on Translation

• Different types of mutations can affect the translation process, leading to altered protein sequences and potentially affecting protein function.

• Normal sequence example: ATG AAC CGT CGC TCA CCG TTA TTG CGT.

  • Protein: Met Asn Arg Arg Ser Pro Leu Leu Arg

• Silent mutation: ATG AAC CGT CGC TCC CCG TTA TTG CGT

  • Protein: Met Asn Arg Arg Ser Pro Leu Leu Arg (no change in amino acid sequence). This is due to the degeneracy of the genetic code.

• Conservative missense mutation: ATG AAC CGT CGC ACA CCG TTA TTG CGT

  • Protein: Met Asn Arg Arg Thr Pro Leu Leu Arg (similar amino acid). The new amino acid has similar properties to the original.

• Nonconservative missense mutation: ATG AAC CGT CGC CCA CCG TTA TTG CGT

  • Protein: Met Asn Arg Arg Pro Pro Leu Leu Arg (different amino acid). The new amino acid has different properties, potentially affecting protein folding and function.

• Nonsense mutation: ATG AAC CGT CGC TAA CCG TTA TTG CGT

  • Protein: Met Asn Arg Arg Stop (premature termination). This leads to a truncated protein.

• Frame-shift mutation: ATG AAC CGT CGC TCG ACC GTT ATT GCG

  • Protein: Met Asn Arg Arg Ser Thr Val Ile Ala (completely altered sequence). This is due to the insertion or deletion of nucleotides, shifting the reading frame.

Mechanism of Translation

The mechanism of translation involves several key steps:

• Activation of amino acids: synthesis of aminoacyl-tRNA. This involves attaching the correct amino acid to its corresponding tRNA molecule.

• Initiation: forming the initiation complex. This involves the assembly of the ribosome, mRNA, and initiator tRNA.

• Elongation: sequential addition of amino acids according to the mRNA sequence. This involves the formation of peptide bonds between amino acids.

• Termination: ending protein synthesis. This occurs when a stop codon is encountered.

• Post-translational modifications: additional modifications to the polypeptide to produce a functional protein. These include folding, glycosylation, and phosphorylation.

Activation of Amino Acids

• This involves the synthesis of aminoacyl-tRNA.

• Process:-

  • ATP + amino acid + tRNA + aminoacyl-tRNA-synthase \rightarrow aminoacyl-tRNA + AMP + PPi

Binding Sites of Ribosomes

• Ribosomes have three binding sites for tRNA:-

  • A (aminoacyl) site: binds the incoming aminoacyl-tRNA.

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

  • E (exit) site: where the tRNA exits after transferring its amino acid.

Initiation

• Involves the formation of the initiation complex.

• Requires:-

  • Ribosome (70S in prokaryotes).

  • Initiation factors (IF1, IF2, IF3 in prokaryotes).

  • Shine-Dalgarno sequence (in prokaryotes) for mRNA binding to ribosome. This sequence is complementary to a region in the 16S rRNA of the 30S subunit.

  • fMet-tRNA (in prokaryotes) or Met-tRNA (in eukaryotes).

  • Start codon AUG.

• Process:-

  • mRNA binds to the 30S ribosomal subunit.

  • fMet-tRNA binds to the start codon in the P site.

  • The 50S subunit joins to form the 70S initiation complex.

Elongation

• Involves the addition of amino acids to the growing polypeptide chain.

• Requires elongation factors (EF-Tu, EF-G).

• Process:

  1. Aminoacyl-tRNA binds to the A site with the help of EF-Tu.

  2. Peptidyl transferase catalyzes the formation of a peptide bond between the amino acid in the A site and the growing polypeptide in the P site.

  3. Translocation: the ribosome moves one codon down the mRNA, moving the tRNA in the A site to the P site and the tRNA in the P site to the E site. This requires EF-G.

Termination

• Occurs when a stop codon (UAA, UAG, UGA) enters the A site.

• Requires release factors (RF-1, RF-2, RF-3).

• Process:

  1. Release factor binds to the stop codon in the A site.

  2. Peptidyltransferase hydrolyzes the bond between the polypeptide and the tRNA in the P site, releasing the polypeptide.

  3. The ribosome dissociates into its 30S and 50S subunits.

Differences in Translation Between Prokaryotes and Eukaryotes

• Ribosomes: Eukaryotes have 80S ribosomes, while prokaryotes have 70S ribosomes.

• Initiation: Eukaryotes use Met as the first amino acid, while prokaryotes use formylmethionine (fMet). Eukaryotes require 11 initiation factors, while prokaryotes require only 3. The initiation complex formation is more complex in eukaryotes.

• Elongation: The elongation process is largely the same in both.

• Termination: Eukaryotes have one release factor, while prokaryotes have two.

Targeting of Synthesized Proteins

• Proteins are targeted to different cellular locations based on signal sequences.

• Examples of signal sequences:-

  • Signal peptide of secretory proteins. This targets proteins to the ER for secretion.

  • Signal peptide of mitochondrial proteins. This targets proteins to the mitochondria.

  • Signal peptide of ER proteins. This retains proteins in the ER.

  • Signal peptide of lysosomal proteins (e.g., Mannose-6-phosphate). This targets proteins to the lysosomes.

Synthesis of Secretory and Membrane Proteins

• Synthesis starts in the cytoplasm.

• Signal Recognition Particle (SRP) binds to the signal peptide and ribosome. SRP is a ribonucleoprotein complex.

• SRP escorts the ribosome to the rough endoplasmic reticulum (RER). The SRP receptor on the ER membrane binds the SRP.

• The protein is translocated into the ER lumen, where it undergoes further processing and modification. This is facilitated by the translocon.

• Signal peptidase cleaves the signal peptide. This removes the signal peptide from the protein.

Inhibitors of Translation

• Various antibiotics and toxins can inhibit translation by targeting different steps in the process.

Modifications of Synthesized Polypeptide

• Modifications can occur co-translationally (during synthesis) or post-translationally (after synthesis).

Cotranslational Modifications

• Deformylation of Met (in prokaryotes, mitochondria, and chloroplasts). This removes the formyl group from methionine.

• Removal of N-terminal amino acids (Met, signal peptide). This can affect protein stability and function.

• Formation of disulfide (S-S) bonds. This stabilizes protein structure.

• Chemical modifications of amino acids (methylation, acylation, hydroxylation, phosphorylation, glycosylation, iodation). These can affect protein activity and interactions.

• Formation of secondary and tertiary structure (folding). This is essential for protein function.

Posttranslational Modifications

• Cut out of peptides (activation, e.g., proinsulin to insulin, pepsinogen to pepsin). This converts inactive precursors into active enzymes or hormones.

• Attachment of prosthetic groups (e.g., heme to globin). This is required for the function of some proteins.

• Formation of quaternary structure. This involves the assembly of multiple polypeptide chains into a functional protein complex.

• Folding of proteins and assembly of protein complexes (chaperones). Chaperones assist in protein folding and prevent aggregation.

• Chemical modifications affecting protein activity (phosphorylation, hydroxylation, γ-carboxylation). These can regulate protein activity and interactions.

Synthesis of Insulin

• Starts in the cytoplasm.

• The signal peptide directs it to the ER-Golgi.

• Cotranslational modifications: cut of signal peptide; formation of S-S bonds.

• Posttranslational modifications: Cut out of c chain, resulting in active insulin.

Disturbances of Translation

• Rare but clinically significant.

• Most important disturbances involve mitochondrial translation (mainly due to mutations of mt-tRNA).

• Affected organs: muscles (weakness, atrophy), heart (decreased output), CNS (different symptoms).

• Manifest as