Gene Expression Notes

Gene Expression Notes

Introduction to Gene Expression

Gene expression is the process by which DNA directs protein synthesis and involves two main stages: transcription and translation. This process aligns with the central dogma of molecular biology, which indicates the flow of information from DNA to messenger RNA (mRNA) to proteins.

Key Processes:

• Transcription: Synthesis of messenger RNA from DNA.

• Translation: Creation of polypeptides (proteins) using information from mRNA.

Transcription vs. Translation

• Transcription is responsible for producing mRNA which serves as a bridge between the genetic information in DNA and the synthesis of proteins.

• Translation occurs on ribosomes where mRNA is read, and amino acids are assembled into polypeptides.

Prokaryotic vs. Eukaryotic Cells

• Bacterial Cells (Prokaryotes): Here, transcription and translation are coupled; as mRNA is synthesized, it can be translated immediately.

• Eukaryotic Cells: Transcription occurs in the nucleus producing pre-mRNA which must undergo further processing before it moves to the cytoplasm for translation.

The Genetic Code

• Codons: The flow of information from gene to protein is achieved through codons, which are tri-nucleotide sequences that code for specific amino acids.

Codons Examples:

1. UGG → Tryptophan

2. UUU → Phenylalanine

Codon Characteristics:

• 64 total codons:

  • 61 code for amino acids.

  • 3 serve as stop signals (UAA, UGA, UAG).

  • 1 start codon (AUG), also codes for Methionine.

• The code is redundant: multiple codons (e.g., UUU and UUC) may code for the same amino acid, showcasing the concept of 'wobble'.

Transcription Process

Transcription consists of three main stages:

1. Initiation

2. Elongation

3. Termination

Detailed Steps of Transcription:

1. Initiation

• Promoter Region: Increase in TATA boxes, critical for RNA polymerase attachment.

• Formation of the transcription initiation complex consisting of:

  • Transcription factors

  • RNA polymerase

2. Elongation

• RNA polymerase synthesizes RNA by unwinding the DNA helix (10-20 bases at a time).

• Progresses at about 40 nucleotides per second, adding nucleotides at the 3’ end to form the growing RNA strand.

3. Termination

• In bacteria, transcription stops at the terminator sequence, while in eukaryotes, it stops after transcribing a poly A signal, releasing the mRNA and requiring modification.

RNA Processing in Eukaryotes

After transcription, the pre-mRNA undergoes processing:

• 5’ Methyl Cap: Added to the 5’ end.

• 3’ Poly A Tail: Added to the 3’ end.

• Introns Removal: Non-coding regions (introns) are cut out and coding segments (exons) are joined together.

Functions of RNA Modifications:

• Facilitate mRNA export from the nucleus.

• Protect mRNA from enzymatic degradation.

• Aid in ribosome attachment for the synthesis of proteins.

Splicing and Ribozymes

• Splicing: Carries through the spliceosome machinery, which recognizes intron-exon boundaries and excises introns, joining exons together.

• Ribozymes: RNA molecules with enzymatic activity (cutting RNA) challenge earlier beliefs that only proteins serve as enzymes.

Functional Importance of Introns

1. Gene Regulation: Introns may contain regulatory sequences that influence gene expression.

2. Alternative Splicing: A single gene can lead to multiple proteins depending on which exons are included in the final mRNA transcript.

3. Exon Shuffling: Introns promote genetic diversity and evolution of proteins by allowing different combinations of exons.

Example of Alternative RNA Splicing:

• A gene can produce several proteins:

  • Protein with all three exons.

  • Protein with exons one and two.

  • Protein with exons two and three.

Summary

Understanding gene expression is integral to comprehending how proteins are constructed from the genetic blueprints within DNA. This knowledge is fundamental in fields such as genetics, molecular biology, and biotechnology.

• Translation is the RNA-directed synthesis of polypeptides, which involves the decoding of messenger RNA (mRNA) to produce proteins.

• The entire process occurs primarily in the ribosome, a complex molecular machine, where mRNA is translated into polypeptides with the indispensable assistance of transfer RNA (tRNA).

Structure of tRNA

• tRNA Characteristics:

  • tRNA is a single-stranded RNA molecule composed of approximately 80 nucleotides, which allows for unique structural conformations.

  • Each tRNA molecule has a specific amino acid attachment site at one end, allowing it to bind the corresponding amino acid, while the anticodon at the other end is crucial for recognition of the matched codon on the mRNA.

  • The flattened 3D structure of tRNA resembles a cloverleaf due to its specific folding, which is essential for its function in translation.

• tRNA Structure:

  • Amino acid attachment site: This site specifically binds to the amino acid corresponding to the tRNA, ensuring the correct amino acid is added during protein synthesis.

  • Anticodon: This three-nucleotide sequence is complementary to the mRNA codon, facilitating accurate pairing and ensuring that the correct amino acid is incorporated into the growing polypeptide chain.

Matching tRNA with Amino Acids

• Two Key Steps:

  1. Correct match between tRNA and amino acid: This critical pairing is catalyzed by the enzyme aminoacyl-tRNA synthetase, which ensures specificity in the translation process.

  2. Correct pairing of the tRNA anticodon with mRNA codon: This ensures the fidelity of protein synthesis, as any mismatch could lead to incorrect amino acid incorporation.

• Amino Acid-tRNA Bonding Process:

  1. The amino acid and tRNA are brought into the active site of the relevant enzyme (e.g., tyrosyl-tRNA synthetase), which recognizes both the tRNA and its corresponding amino acid.

  2. The hydrolysis of ATP provides energy required to catalyze the accurate formation of the ester bond between the amino acid and the tRNA.

  3. Once the amino acid is covalently attached to the tRNA, the aminoacyl-tRNA is released, ready for delivery to the ribosome for polypeptide synthesis.

Structure of Ribosomes

• Ribosome Function: The ribosome is essential for protein synthesis as it facilitates the coupling of tRNA anticodons with their corresponding mRNA codons, enabling the translation of genetic information into functional proteins.

• Prokaryotic vs Eukaryotic Ribosomes:

  • While both types share basic structural similarities, eukaryotic ribosomes are generally larger and more complex than prokaryotic ribosomes.

  • Eukaryotic ribosomes consist of:

  • 80 ribosomal proteins.

  • 4 ribosomal RNAs (rRNAs): 18S, 5S, 5.8S, and 28S.

  • Prokaryotic ribosomes typically have fewer ribosomal proteins and rRNAs, reflecting their fundamentally different cellular environments.

• Ribosome Composition:

  • Large subunit: Comprised of 50 ribosomal proteins along with 3 rRNAs (28S, 5S, and 5.8S), which play crucial roles in the structure and function of the ribosome.

  • Small subunit: Contains 30-40 ribosomal proteins and one rRNA (18S).

  • Binding Sites:

  • A site (Aminoacyl site): Where the incoming aminoacyl-tRNA binds, adding its amino acid to the growing peptide chain.

  • P site (Peptidyl site): Holds the tRNA carrying the growing polypeptide chain.

  • E site (Exit site): Where discharged tRNA exits the ribosome after delivering its amino acid.

Stages of Translation

1. Initiation:

   • The formation of the translation initiation complex at the P site is essential for starting the protein synthesis. This complex is composed of initiator tRNA (which carries methionine), mRNA, the small ribosomal subunit, and various initiation factors.

   • The ribosome recognizes the start codon AUG on the mRNA, often guided by the Shine-Dalgarno sequence in prokaryotes, which helps position the ribosome correctly at the beginning of the coding region.

2. Elongation:

   • The addition of amino acids occurs one at a time, facilitated by elongation factors that assist the tRNA-mRNA pairing.

   • This step involves three main processes: codon recognition (where the correct tRNA pairs with the mRNA codon), peptide bond formation (catalyzed by the ribosome’s peptidyl transferase activity), and translocation of the ribosome along the mRNA in the 5' to 3' direction to expose the next codon.

3. Termination:

   • This phase is triggered when a stop codon (UAG, UAA, UGA) is encountered.

   • A release factor enters the A site, leading to the addition of water instead of an amino acid, which facilitates the release of the finished polypeptide from the tRNA.

   • Subsequently, the ribosomal subunits dissociate from the mRNA, completing the translation process, and allowing for the potential recycling of ribosomal components and tRNA.

Post-Translation Modifications

• After the synthesis of proteins, many undergo critical modifications, including folding, which often involves forming complex tertiary or quaternary structures essential for their functionality.

• Protein Folding:

  • The linear polypeptide chain acquires its specific functional form through folding. Molecular chaperones often assist in this process, ensuring the correct conformation is achieved.

• Signal Peptides:

  • These are short peptides, approximately 16 amino acids long, which tag polypeptides destined for the endoplasmic reticulum (ER).

  • Free ribosomes synthesize cytosolic proteins, while ribosomes attached to the ER synthesize proteins that will be secreted or inserted into membranes. The targeting process to the ER involves:

  1. The Signal Recognition Particle (SRP) binds to the signal peptide as synthesis is ongoing.

  2. The SRP-receptor then allows the complex to dock onto the ER membrane.

  3. Synthesis resumes after the SRP detachment, and the signal peptide is cleaved off post-synthesis.

• Polyribosomes (Polysomes):

  • Multiple ribosomes can simultaneously translate a single mRNA strand, forming a polysome, which greatly speeds up protein production by allowing many identical polypeptides to be synthesized in parallel.

Coupling of Transcription and Translation

• In prokaryotic organisms, transcription and translation are coupled; as soon as mRNA is transcribed, ribosomes can bind and begin translation immediately due to the lack of nuclear membranes.

• In eukaryotes, these processes occur in separate cellular compartments: transcription takes place in the nucleus, and translation occurs in the cytoplasm, thus creating a spatial separation that necessitates additional processing of mRNA before it can be translated.

Mutations in Protein Synthesis

• Mutations can critically alter both the structure and function of proteins, which can have significant implications for cellular function.

• Point Mutations: These are defined as changes occurring in a single base pair of DNA, leading, in turn, to potential changes in the encoded amino acid sequence.

  • Point mutations can yield various effects, such as those observed in sickle cell anemia, which is a classic example of a missense mutation.

  • Categories of Point Mutations:

    • Substitutions: These can result in silent (no change to amino acid), missense (change to amino acid), or nonsense (premature stop) mutations.

    • Insertions/Deletions: Adding or removing nucleotides can lead to frameshift mutations, which drastically alter the downstream amino acid sequence.

• Example of an Effect of Mutation:

  • Sickle Cell Anemia: Caused by a mutation that changes an adenine (A) to a thymine (T), leading to the substitution of valine for glutamic acid in the protein hemoglobin, which significantly alters its structure and function, resulting in the characteristic sickle-shaped red blood cells.

• Translation is the RNA-directed synthesis of polypeptides, which involves the decoding of messenger RNA (mRNA) to produce proteins.

• The entire process occurs primarily in the ribosome, a complex molecular machine, where mRNA is translated into polypeptides with the indispensable assistance of transfer RNA (tRNA).

Structure of tRNA

• tRNA Characteristics:

  • tRNA is a single-stranded RNA molecule composed of approximately 80 nucleotides, which allows for unique structural conformations.

  • Each tRNA molecule has a specific amino acid attachment site at one end, allowing it to bind the corresponding amino acid, while the anticodon at the other end is crucial for recognition of the matched codon on the mRNA.

  • The flattened 3D structure of tRNA resembles a cloverleaf due to its specific folding, which is essential for its function in translation.

• tRNA Structure:

  • Amino acid attachment site: This site specifically binds to the amino acid corresponding to the tRNA, ensuring the correct amino acid is added during protein synthesis.

  • Anticodon: This three-nucleotide sequence is complementary to the mRNA codon, facilitating accurate pairing and ensuring that the correct amino acid is incorporated into the growing polypeptide chain.

Matching tRNA with Amino Acids

• Two Key Steps:

  1. Correct match between tRNA and amino acid: This critical pairing is catalyzed by the enzyme aminoacyl-tRNA synthetase, which ensures specificity in the translation process.

  2. Correct pairing of the tRNA anticodon with mRNA codon: This ensures the fidelity of protein synthesis, as any mismatch could lead to incorrect amino acid incorporation.

• Amino Acid-tRNA Bonding Process:

  1. The amino acid and tRNA are brought into the active site of the relevant enzyme (e.g., tyrosyl-tRNA synthetase), which recognizes both the tRNA and its corresponding amino acid.

  2. The hydrolysis of ATP provides energy required to catalyze the accurate formation of the ester bond between the amino acid and the tRNA.

  3. Once the amino acid is covalently attached to the tRNA, the aminoacyl-tRNA is released, ready for delivery to the ribosome for polypeptide synthesis.

Structure of Ribosomes

• Ribosome Function: The ribosome is essential for protein synthesis as it facilitates the coupling of tRNA anticodons with their corresponding mRNA codons, enabling the translation of genetic information into functional proteins.

• Prokaryotic vs Eukaryotic Ribosomes:

  • While both types share basic structural similarities, eukaryotic ribosomes are generally larger and more complex than prokaryotic ribosomes.

  • Eukaryotic ribosomes consist of:

  • 80 ribosomal proteins.

  • 4 ribosomal RNAs (rRNAs): 18S, 5S, 5.8S, and 28S.

  • Prokaryotic ribosomes typically have fewer ribosomal proteins and rRNAs, reflecting their fundamentally different cellular environments.

• Ribosome Composition:

  • Large subunit: Comprised of 50 ribosomal proteins along with 3 rRNAs (28S, 5S, and 5.8S), which play crucial roles in the structure and function of the ribosome.

  • Small subunit: Contains 30-40 ribosomal proteins and one rRNA (18S).

  • Binding Sites:

  • A site (Aminoacyl site): Where the incoming aminoacyl-tRNA binds, adding its amino acid to the growing peptide chain.

  • P site (Peptidyl site): Holds the tRNA carrying the growing polypeptide chain.

  • E site (Exit site): Where discharged tRNA exits the ribosome after delivering its amino acid.

Stages of Translation

1. Initiation:

   • The formation of the translation initiation complex at the P site is essential for starting the protein synthesis. This complex is composed of initiator tRNA (which carries methionine), mRNA, the small ribosomal subunit, and various initiation factors.

   • The ribosome recognizes the start codon AUG on the mRNA, often guided by the Shine-Dalgarno sequence in prokaryotes, which helps position the ribosome correctly at the beginning of the coding region.

2. Elongation:

   • The addition of amino acids occurs one at a time, facilitated by elongation factors that assist the tRNA-mRNA pairing.

   • This step involves three main processes: codon recognition (where the correct tRNA pairs with the mRNA codon), peptide bond formation (catalyzed by the ribosome’s peptidyl transferase activity), and translocation of the ribosome along the mRNA in the 5' to 3' direction to expose the next codon.

3. Termination:

   • This phase is triggered when a stop codon (UAG, UAA, UGA) is encountered.

   • A release factor enters the A site, leading to the addition of water instead of an amino acid, which facilitates the release of the finished polypeptide from the tRNA.

   • Subsequently, the ribosomal subunits dissociate from the mRNA, completing the translation process, and allowing for the potential recycling of ribosomal components and tRNA.

Post-Translation Modifications

• After the synthesis of proteins, many undergo critical modifications, including folding, which often involves forming complex tertiary or quaternary structures essential for their functionality.

• Protein Folding:

  • The linear polypeptide chain acquires its specific functional form through folding. Molecular chaperones often assist in this process, ensuring the correct conformation is achieved.

• Signal Peptides:

  • These are short peptides, approximately 16 amino acids long, which tag polypeptides destined for the endoplasmic reticulum (ER).

  • Free ribosomes synthesize cytosolic proteins, while ribosomes attached to the ER synthesize proteins that will be secreted or inserted into membranes. The targeting process to the ER involves:

  1. The Signal Recognition Particle (SRP) binds to the signal peptide as synthesis is ongoing.

  2. The SRP-receptor then allows the complex to dock onto the ER membrane.

  3. Synthesis resumes after the SRP detachment, and the signal peptide is cleaved off post-synthesis.

• Polyribosomes (Polysomes):

  • Multiple ribosomes can simultaneously translate a single mRNA strand, forming a polysome, which greatly speeds up protein production by allowing many identical polypeptides to be synthesized in parallel.

Coupling of Transcription and Translation

• In prokaryotic organisms, transcription and translation are coupled; as soon as mRNA is transcribed, ribosomes can bind and begin translation immediately due to the lack of nuclear membranes.

• In eukaryotes, these processes occur in separate cellular compartments: transcription takes place in the nucleus, and translation occurs in the cytoplasm, thus creating a spatial separation that necessitates additional processing of mRNA before it can be translated.

Mutations in Protein Synthesis

• Mutations can critically alter both the structure and function of proteins, which can have significant implications for cellular function.

• Point Mutations: These are defined as changes occurring in a single base pair of DNA, leading, in turn, to potential changes in the encoded amino acid sequence.

  • Point mutations can yield various effects, such as those observed in sickle cell anemia, which is a classic example of a missense mutation.

  • Categories of Point Mutations:

    • Substitutions: These can result in silent (no change to amino acid), missense (change to amino acid), or nonsense (premature stop) mutations.

    • Insertions/Deletions: Adding or removing nucleotides can lead to frameshift mutations, which drastically alter the downstream amino acid sequence.

• Example of an Effect of Mutation:

  • Sickle Cell Anemia: Caused by a mutation that changes an adenine (A) to a thymine (T), leading to the substitution of valine for glutamic acid in the protein hemoglobin, which significantly alters its structure and function, resulting in the characteristic sickle-shaped red blood cells.