Protein Synthesis | Medical Science Optional Notes for UPSC PDF Download

Protein Synthesis-Repeats

• Explain the process of protein synthesis, including the identification of inhibitors and their respective sites of action (1997).
• Provide a flow chart illustrating the synthesis of mRNA in eukaryotic cells, and elucidate the posttranscriptional modifications involved (2001).
• Explore the divergence between the initially synthesized polypeptide chain and the final protein molecule essential for cellular function. Illustrate how modifications are made to the chain to confer biological activity, citing relevant examples (2006).
• Elaborate on the precise molecular mechanism governing the initiation and termination of polypeptide synthesis on ribosomes. Discuss the consequences of a failed termination event in your own words (2009).
• Outline the key components of the translation apparatus in eukaryotic cells (2011).
• Provide insights into the post-translational modifications of proteins, offering a comprehensive note on the subject (2012).
• Provide a detailed description of the structure of tRNA, and discuss the energetics involved in the synthesis of a single peptide bond. Additionally, identify inhibitors of protein synthesis (2018).

Central Dogma of Life

Protein synthesis is the mechanism that converts the nucleotide triplets, known as codons, in messenger RNA (mRNA) into the amino acid code, comprising 20 symbols, responsible for constructing the polypeptide chains forming proteins. This synthesis occurs through two sequential steps: transcription and translation.

In transcription, the genetic information stored in DNA is transcribed into mRNA. Subsequently, the mRNA exits the cell's nucleus and enters the cytoplasm. During translation, the mRNA collaborates with ribosomes and transfer RNA (tRNA) to orchestrate the synthesis of proteins.

Transcription

Transcription-in Brief

• The initial step in transcription, involves partially unwinding the DNA molecule. This allows access to the specific portion of DNA that encodes the necessary protein for transcription.
• Following the correct unwinding of the DNA molecule, an enzyme known as RNA polymerase assists in aligning nucleotides to form a complementary mRNA strand.
• As mRNA is a single-stranded molecule, only one of the two DNA strands serves as a template for the synthesis of the new RNA strand.

Transcription in Prokaryotes

• The attachment of the RNA polymerase enzyme to DNA.
• The promoter region, a distinct segment on DNA, serves as the site where the enzyme binds.
• On the coding strand, there are two fundamental sequences recognized by the sigma factor of RNA polymerase:
  i) The Pribnow box (TATA box) with the sequence TATAAT.
  ii) The '-35' sequence with the sequence TTGACA.
• Release of sigma factor 
• RNA is synthesized from 5' end to 3' end antiparallel to the DNA template

There are two distinct types of terminations in transcription:

• Rho-dependent termination involves the binding of the rho factor to the advancing RNA, leading to termination and release of the RNA. It also facilitates the dissociation of RNA polymerase from DNA.
• Rho-independent termination occurs through the creation of hairpin structures resulting from palindromic sequences, promoting complementary base pairing.

The prokaryotic mRNA synthesized transcription is almost similar to functional mRNA

Transcription in Eukaryotes

Initiation is a compli process

• There are three distinct polymerases (I, II, III).
• RNA polymerase II is responsible for synthesizing precursors for mRNAs and is commonly known as hnRNA (Heterogeneous nuclear RNA).
• Initiation sites include the CAAT box and Hogness box (TATA box).
• Numerous transcription factors, such as TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH, interact with eukaryotic promoter regions.
• Activators are formed when enhancers bind to transcription factors.
• Termination does not require stem-loop structures.
• It undergoes extensive post-transcriptional modifications.

Post Transcriptional Modification

Post-transcription processings required idconvert primary transcript into functional RNAs

• Cleavage: Larger RNA precursors are cleaved to form smaller RNAs. The primary transcript is cleaved by Ribonuclease-P (an RNA enzyme) to produce 5-7 tRNA precursors.
• Capping and Tailing: Initially, at the 5' end, a 7-methylguanosine cap (7mG) is added, and at the 3' end, a poly-A tail is attached. The cap is a chemically modified molecule of guanosine triphosphate (GTP).
• Splicing: Eukaryotic primary mRNAs consist of non-coding introns and coding exons. The introns are removed through RNA splicing, a process involving ATP to cut the RNA, releasing introns and connecting adjacent exons to generate mature mRNA.
• Nucleotide Modifications: Common modifications include tRNA methylation (e.g., methyl cytosine, methyl guanosine), deamination (e.g., inosine from adenine), dihydrouracil, pseudouracil, etc.

Translation

• Translation initiates with the binding of the mRNA strand to the ribosome.
• The first codon, always the start codon methionine, occupies the P site, while the second codon occupies the A site.
• The tRNA molecule, with an anticodon complementary to the mRNA, forms a temporary base pair with the mRNA in the A site.
• A peptide bond is established between the amino acid attached to the tRNA in the A site and the methionine in the P site.
• The ribosome shifts along the mRNA, causing the tRNA in the A site to move to the P site, with a new codon filling the A site.
• The suitable tRNA carrying the corresponding amino acid forms base pairs with the newly introduced codon in the A site.
• Another peptide bond is formed between the two adjacent amino acids held by tRNA molecules.

• The ribosome undergoes another shift, releasing the tRNA from the P site into the cytoplasm, where it will eventually bind with another amino acid.
• Another tRNA arrives to pair with the recently introduced codon in the A site, leading to the formation of a peptide bond between the new amino acid and the extending peptide chain.

The process continues until one of the three stop codons enters the A site. At that point, the protein chain connected to the tRNA in the P site is released.

The proteins produced during translation are initially non-functional. Subsequent to the completion of protein synthesis, numerous modifications occur in the polypeptides. These modifications encompass:

1. Folding of Proteins
The three-dimensional structure of proteins plays a crucial role in their biological activities.
A significant number of proteins achieve their proper conformation with the assistance of specific proteins known as chaperones.
Chaperones, also recognized as heat shock proteins (HSP), play a vital role in guiding and promoting interactions on the surfaces of polypeptides, ultimately leading to the specific conformation of a protein. Additionally, they contribute to stabilizing the folding process and preventing undesired conformations.

Types of Chaperones

Disorders of protein misfolding
Cystic fibrosis (CF) is an autosomal recessive disorder resulting from a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
This results in the production of a modified protein due to misfolding. The misfolded protein undergoes rapid degradation.

Prions, which are proteinaceous infectious agents, are misfolded proteins linked to various fatal neurodegenerative diseases in both animals and humans.

individuals and animals in good health. However, the PrP found in infectious material exhibits a distinct structure and is resistant to proteases, the body's enzymes that typically break down proteins.

2. Degradation By Proteolysis
Precursor proteins undergo a process known as trimming to release active proteins. The transformation of preproinsulin into insulin serves as a classic example.

3. Intein Splicing
Inteins are intervening sequences within certain proteins that must be excised for the protein to become active. These are analogous to introns in mRNAs. Exteins are akin to exons in mRNAs and are subsequently ligated after intein splicing.

4. Covalent Modifications
Through alterations in amino acids, proteins can undergo conversion into either an active or inactive form. Specific examples of covalent modifications are outlined below.

• Phosphorylation: Protein kinase facilitates phosphorylation, a process that can either enhance or diminish enzymatic activity.
• Hydroxylation: In collagen formation, the amino acids proline and lysine transform into hydroxyproline and hydroxylysine, respectively. Vitamin C plays a crucial role in this hydroxylation reaction.
• Carboxylation: Vitamin K-dependent carboxylation occurs in glutamic acid residues of certain clotting factors (vitamin K-dependent clotting factors II, VII, IX, and X, as well as proteins C, S, and Z). Notably, the anticoagulant Warfarin reduces blood clotting by inhibiting the enzyme vitamin K epoxide reductase, hindering the reactivation of vitamin K. Consequently, clotting factors II, VII, IX, and X exhibit decreased clotting ability.
• Glycosylation: The attachment of a carbohydrate moiety is crucial for enabling some proteins to carry out their functions.

Transfer RNA

Inhibitors of Protein Synthesis

Bacterial protein synthesis

Energetics of peptide bond formation

In the course of translation, a crucial step known as the formation of a peptide bond takes place, linking two amino acids to construct the protein's backbone. The enzyme responsible for this is peptidyl transferase. Notably, the formation of a peptide bond itself does not demand energy.

During protein synthesis, tRNA facilitates the transportation of amino acids. Prior to attachment to tRNA, these amino acids undergo activation. Aminoacyl RNA synthetase is the enzyme engaged in the activation process, which necessitates the use of ATP. However, since the already activated amino acids participate in the formation of peptide bonds, this specific process does not require additional ATP.