Gene Transcription and Translation: Biochemistry Study Notes

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Gene Transcription and Translation

Overview

This section covers the molecular processes of gene transcription and translation, which are central to the flow of genetic information from DNA to protein in cells. Understanding these mechanisms is essential for biochemistry students, as they underpin gene expression, regulation, and protein synthesis.

Transcription

Definition and Process

Transcription: The synthesis of RNA from a DNA template, catalyzed by RNA polymerase.
Enzymes: RNA polymerases generate a single-stranded RNA molecule.

Directionality and Sequence

The DNA template is read in the 3'-to-5' direction, while RNA is synthesized in the 5'-to-3' direction.
The RNA transcript is complementary to the DNA template strand and matches the coding strand (except U replaces T).

RNA Polymerase

Function: Synthesizes RNA by adding ribonucleotides to the growing chain.
Prokaryotes: Use a single RNA polymerase.
Eukaryotes: Have three main types (I, II, III) with specific roles.
RNA polymerases can initiate synthesis without a primer, unlike DNA polymerases.

Polymerase	Product
RNA polymerase I	rRNA (except 5S)
RNA polymerase II	mRNA, snRNA
RNA polymerase III	tRNA, 5S rRNA

Promoter Location and Core Promoter

Upstream of the Gene: Promoter for RNA polymerase II is located upstream (5' direction) of the transcription start site (TSS).
Core Promoter: Contains essential elements (often within 50-100 bp upstream of TSS) for RNA polymerase II binding and transcription initiation.

Initiation

Promoter Binding: RNA polymerase and transcription factors bind to the promoter region (e.g., TATA box).
Transcription Initiation Complex: Assembly of RNA polymerase and proteins (including TATA-binding protein, TBP) at the promoter.

Promoter Strength

Core Promoter Elements: Initiator (Inr) element affects transcription start and promoter strength.
Proximal Promoter Elements: CAAT box, GC-rich regions enhance activity by recruiting transcription factors.
Binding Affinity: Strong promoters have multiple transcription factor binding sites, increasing RNA polymerase recruitment.
Coactivators/Enhancers: Interact with promoters to boost transcriptional activity.

Elongation

RNA Synthesis: RNA polymerase moves along DNA, synthesizing RNA in the 5'-to-3' direction.
RNA Nucleotides: Complementary ribonucleotides are added to the growing RNA strand.

Termination

Termination Signal: Specific DNA sequences (e.g., polyadenylation signals) signal the end of transcription.
Release of RNA: Newly synthesized RNA is released, and RNA polymerase dissociates from DNA.

Post-Transcriptional Modifications

5' Capping

Purpose: Protects mRNA from degradation and assists in ribosome binding during translation.

Polyadenylation (Poly-A Tail)

Purpose: Addition of adenine nucleotides to the 3' end, enhancing mRNA stability.

Splicing (Removal of Introns)

Introns and Exons: Introns (non-coding) are removed; exons (coding) are joined.
Spliceosome: Protein-RNA complex that carries out splicing.

Differences Between Prokaryotic and Eukaryotic Transcription

Feature	Eukaryotes (Human)	Prokaryotes (E. coli)
Nucleus	Yes	No
Chromosomes	23 pairs (linear)	1 (circular)
Histones	Yes	No
Genes	Unique	100%
Polycistronic mRNA	No	Yes
Introns	Yes	No
Transcription/Translation	Separate (nucleus/cytoplasm)	Coupled

Ribosomal Synthesis

rRNA Transcription

Location: Occurs in the nucleolus.
rRNA Genes: Transcribed by RNA polymerase I (45S pre-rRNA) and III (5S rRNA).
Primary Transcript: 45S pre-rRNA contains 18S, 5.8S, and 28S rRNA sequences.

Ribosome Assembly

Small Subunit (40S): Contains 18S rRNA and proteins.
Large Subunit (60S): Contains 5S, 5.8S, and 28S rRNAs and proteins.
Pre-Ribosomal Particles: Assembled in nucleolus, mature in cytoplasm.

Synthesis and Structure of tRNA

Synthesis of tRNA

RNA Polymerase III: Transcribes tRNA genes in the nucleus.
Promoter Elements: Internal sequences guide transcription initiation.
Primary Transcript: Precursor tRNA (pre-tRNA) undergoes processing.

Structures of tRNA

D-Loop: Contains modified bases, important for folding and aminoacylation.
Anticodon Loop: Contains anticodon, pairs with mRNA codon.
TψC Loop: Contains modified bases, interacts with ribosome.
3' CCA End: Site for amino acid attachment, essential for translation.

Types of tRNA

Type of tRNA	Function	Example
Canonical tRNA	Carries standard amino acids	tRNAPhe, tRNALeu
Initiator tRNA	Recognizes start codon	tRNAMet
Elongator tRNA	Elongation phase	tRNA for other amino acids
tRNA for Selenocysteine	Incorporates selenocysteine	tRNASec
tRNA for Pyrrolysine	Incorporates pyrrolysine	tRNAPyl
Suppression tRNA	Recognizes stop codons	Modified tRNAGln

Aminoacyl-tRNA Synthetases

Activation of Amino Acid: Amino acid reacts with ATP to form aminoacyl-AMP, releasing pyrophosphate (PPi).

Transfer to tRNA: Aminoacyl-AMP is transferred to tRNA's 3'-OH group, forming aminoacyl-tRNA and releasing AMP.

Specificity: Each amino acid is attached by a specific aminoacyl-tRNA synthetase.

Codon and Anticodon

Codon: Sequence of three nucleotides on mRNA specifying an amino acid (e.g., AUG for methionine).
Anticodon: Sequence of three nucleotides on tRNA, complementary to mRNA codon (e.g., UAC on tRNAMet pairs with AUG).

The Genetic Code

Total Codons: 64
Stop Codons: UGA, UAG, UAA (terminate translation)
Sense Codons: 61 codons specify amino acids
Some amino acids have only one codon (AUG for methionine, UGG for tryptophan).

First Base	Second Base	Third Base
A	U	G
U	A	A
G	G	G
...	...	...

Mutations

Definition: Change in DNA sequence affecting gene function.
Importance: Can alter protein structure/function, cause disease, or contribute to genetic diversity.

Type	Description	Example
Point	Single base change	A → G
Silent	No amino acid change	GAA → GAG
Missense	Changes amino acid	GAG → GTG
Nonsense	Produces stop codon	CGA → UGA
Insertion	Adds bases	CAG repeats
Deletion	Removes bases	ΔF508
Frameshift	Shifts reading frame	HEXA gene

Types of Mutations

Substitution: One nucleotide replaced by another (e.g., sickle cell anemia).
Silent Mutation: No change in amino acid sequence.
Missense Mutation: Changes one amino acid.
Nonsense Mutation: Introduces premature stop codon.
Insertion: Adds nucleotides (e.g., Huntington's disease).
Deletion: Removes nucleotides (e.g., cystic fibrosis).
Frameshift Mutation: Alters reading frame, often leading to nonfunctional protein (e.g., Tay-Sachs disease).

Translation

Overview

Translation is the process by which the genetic code in mRNA is decoded to synthesize proteins. It occurs in the ribosome and involves initiation, elongation, and termination steps.

Initiation

mRNA Binding: Small ribosomal subunit binds mRNA near start codon (AUG).
Initiator tRNA: tRNAMet pairs with AUG.
Initiation Complex: Large subunit joins, forming complete ribosome; initiator tRNA in P site.
EPA Sites: P (peptidyl), A (aminoacyl), E (ejection).

Elongation

Codon Recognition: Aminoacyl-tRNA enters A site, pairs with mRNA codon.
Peptide Bond Formation: Ribosome catalyzes bond between amino acids in P and A sites.
Translocation: Ribosome moves along mRNA, shifting tRNAs through A, P, and E sites.

Termination

Stop Codon Recognition: Ribosome encounters stop codon (UAA, UAG, UGA); release factors bind A site.
Release of Polypeptide: Polypeptide chain released from tRNA in P site; ribosome disassembles.

Post-Translational Modifications

Phosphorylation

Definition: Addition of phosphate group (PO43-) to serine, threonine, or tyrosine residues.
Functions: Regulates protein activity, signal transduction.
Example: Phosphorylation of p53 regulates DNA repair and cell cycle.

Glycosylation

Definition: Addition of carbohydrate groups to proteins (in ER and Golgi).
Functions: Protein folding/stability, cell-cell recognition.
Example: Glycosylation of antibodies for immune function.

Measuring Glycated Hemoglobin (A1C)

A1C Measurement: Percentage of hemoglobin attached to glucose; reflects average blood glucose over 2-3 months.
Normal Range: Below 5.7%.
Prediabetes: 5.7%–6.4%.
Diabetes: 6.5% or higher.
Monitoring: Used to track blood sugar control in diabetes.

Acetylation

Definition: Addition of acetyl group (CH3CO) to lysine residues.
Enzymes: Histone acetyltransferases (HATs) add, histone deacetylases (HDACs) remove acetyl groups.
Functions: Regulates gene expression by altering chromatin structure.

Ubiquitination

Definition: Addition of ubiquitin to lysine residues, tagging proteins for degradation.
Functions: Protein degradation, regulation of cell cycle and immune responses.
Steps: Activation (E1), conjugation (E2), ligation (E3).
Ubiquitin binds to lysine, cysteine, serine, threonine, or N-terminus via peptide, thioester, or ester bonds.

Methylation

Definition: Addition of methyl group (CH3) to lysine or arginine residues.
Enzymes: Methyltransferases add, demethylases remove methyl groups.
Functions: Gene regulation, signal transduction.
Example: Methylation of histone H3 on lysine 9 (H3K9) leads to gene silencing.

Fatty Acylation

Definition: Attachment of lipid molecules to proteins, targeting them to membranes.
Types: Prenylation (farnesyl/geranylgeranyl), palmitoylation (palmitic acid), GPI anchoring.
Functions: Membrane targeting, signal transduction.
Example: Ras proteins are prenylated for plasma membrane association.

Gamma-Carboxylation

Definition: Addition of carboxyl group to gamma carbon of glutamic acid, catalyzed by gamma-glutamyl carboxylase (requires vitamin K).
Biological Roles:
- Blood coagulation: Enables calcium binding for clotting factors.
- Bone metabolism: Facilitates calcium binding for bone mineralization.

Summary Table: Key Processes and Modifications

Process	Key Enzyme	Direction	Main Product
Transcription	RNA polymerase	5' to 3'	mRNA, rRNA, tRNA
Translation	Ribosome	N-terminus to C-terminus	Protein
Phosphorylation	Kinase	---	Phosphorylated protein
Glycosylation	Glycosyltransferase	---	Glycoprotein
Acetylation	HAT/HDAC	---	Acetylated protein
Ubiquitination	E1/E2/E3	---	Ubiquitinated protein
Methylation	Methyltransferase	---	Methylated protein
Fatty Acylation	Acyltransferase	---	Lipidated protein
Gamma-Carboxylation	Gamma-glutamyl carboxylase	---	Carboxylated protein

Additional info: These notes expand on the original slides by providing definitions, examples, and context for each process and modification, ensuring a comprehensive and self-contained study guide for biochemistry students.