Transcription and Translation

Dr. Arvind Kumar

Introduction

The flow of genetic information in all living organisms follows the central dogma of molecular biology, which states that DNA → RNA → Protein.

  • Transcription is the process of copying genetic information from DNA into RNA.

  • Translation is the process of converting the RNA sequence into a specific protein.

  • These processes ensure that genetic information stored in DNA is expressed as functional proteins, which carry out structural and metabolic roles in the cell.

  • Regulation of transcription and translation is essential for proper growth, development, and adaptation of organisms.

Factors Involved in Transcription and Translation

In Transcription

  • DNA Template: Provides the information for RNA synthesis.

  • RNA Polymerase: Enzyme that synthesizes RNA from DNA template.

  • Promoter Region: DNA sequence where RNA polymerase binds to initiate transcription.

  • Transcription Factors (in eukaryotes): Proteins that help RNA polymerase recognize promoter and regulate gene expression.

  • Ribonucleotides (ATP, GTP, UTP, CTP): Building blocks of RNA.

  • Regulatory Sequences: Enhancers, silencers, and operators that control transcription.

In Translation

  • mRNA: Carries the genetic code from DNA to ribosome.

  • Ribosome: Site of protein synthesis, made of rRNA and proteins.

  • tRNA: Brings amino acids to ribosome according to codon sequence.

  • Amino Acids: Building blocks of proteins.

  • Enzymes (Aminoacyl-tRNA synthetase): Attach correct amino acid to its tRNA.

  • Initiation, Elongation, and Release Factors: Proteins that control different stages of translation.

  • GTP/ATP: Energy sources for translation process.

 

RNA Processing 

  • In eukaryotic cells, the RNA formed directly after transcription is called the primary transcript (pre-mRNA).
  • It is not functional and undergoes several modifications to become mature mRNA that can be translated into a protein.

1. Capping (5’ Cap Formation)

  • A 7-methylguanosine (m7G) cap is added to the 5’ end of the pre-mRNA.

  • Functions:

    • Protects mRNA from degradation by exonucleases.

    • Helps in ribosome recognition and initiation of translation.

2. Polyadenylation (Poly-A Tail Addition)

  • At the 3’ end, a chain of adenine nucleotides (Poly-A tail) is added by poly(A) polymerase.

  • Functions:

    • Increases stability of mRNA.

    • Facilitates transport of mRNA from nucleus to cytoplasm.

    • Helps in efficient translation.

3. Splicing

  • Eukaryotic genes contain introns (non-coding regions) and exons (coding regions).

  • Splicing removes introns and joins exons to form a continuous coding sequence.

  • Carried out by a complex called spliceosome (made of snRNA + proteins).

  • Alternative splicing: A single gene can give rise to different proteins by joining exons in different combinations.

4. RNA Editing (in some cases)

  • Nucleotides of RNA may be inserted, deleted, or chemically modified.

  • Changes the coding information.

  • Example: ApoB100 → ApoB48 editing in humans.

 

Types of RNA

  • mRNA (Messenger RNA): Carries genetic code from DNA to ribosomes.

  • tRNA (Transfer RNA): Brings specific amino acids during translation.

  • rRNA (Ribosomal RNA): Structural and catalytic component of ribosomes.

  • snRNA (Small nuclear RNA): Involved in splicing of pre-mRNA.

  • miRNA (Micro RNA) & siRNA (Small interfering RNA): Regulate gene expression by silencing or degrading mRNA.

  • lncRNA (Long non-coding RNA): Regulatory functions in gene expression.

Genetic Code

  • The genetic code is the set of rules by which the sequence of nucleotides in mRNA is translated into the sequence of amino acids in a protein.
  • It is universal for almost all organisms and ensures that the genetic information in DNA is correctly expressed as proteins.

Features of the Genetic Code

  1. Triplet Code

    • Each amino acid is encoded by a sequence of three nucleotides (codon).

    • Example: AUG codes for Methionine.

  2. Universal

    • The same codon codes for the same amino acid in almost all living organisms (bacteria to humans).

  3. Degenerate (Redundant)

    • Most amino acids are coded by more than one codon.

    • Example: Leucine is coded by six codons (UUA, UUG, CUU, CUC, CUA, CUG).

  4. Unambiguous

    • Each codon specifies only one amino acid.

  5. Start Codon

    • AUG is the start codon and also codes for Methionine (initiates translation).

  6. Stop Codons

    • UAA, UAG, UGA → Do not code for any amino acid.

    • They signal termination of protein synthesis.

  7. Non-overlapping and Commaless

    • Codons are read one after another, without overlapping or punctuation.

Types of Codons

  • Sense codons: 61 codons that specify amino acids.

  • Nonsense codons: 3 stop codons (UAA, UAG, UGA).

 

Lac Operon

  • The lac operon is a gene regulatory system in Escherichia coli (E. coli) that controls the metabolism of lactose.
  • It is a classic example of an inducible operon, meaning it is usually OFF but can be switched ON in the presence of lactose.

Components of Lac Operon

  1. Structural Genes

    • lacZ → codes for β-galactosidase (breaks lactose into glucose + galactose).

    • lacY → codes for permease (transports lactose into the cell).

    • lacA → codes for transacetylase (detoxifies by-products).

  2. Regulatory Elements

    • Promoter (P): Site where RNA polymerase binds to start transcription.

    • Operator (O): Site where the repressor binds to block transcription.

    • Regulator Gene (lacI): Produces repressor protein that controls the operon.

Working of Lac Operon

  • Without Lactose (Operon OFF):

    • Repressor protein binds to operator.

    • RNA polymerase cannot move forward.

    • No transcription of structural genes.

  • With Lactose (Operon ON):

    • Lactose is converted into allolactose (inducer).

    • Allolactose binds to repressor → inactivates it.

    • RNA polymerase binds promoter and transcribes structural genes.

    • Enzymes are produced for lactose metabolism.

Regulation by Glucose (Catabolite Repression)

  • When glucose is present, the lac operon remains mostly OFF even if lactose is available.

  • cAMP-CAP complex is required for efficient transcription.

    • Low glucose → High cAMP → cAMP binds CAP → enhances transcription.

    • High glucose → Low cAMP → CAP does not bind → transcription reduced.

 

Tryptophan (Trp) Operon

  • The tryptophan operon is a gene regulatory system in Escherichia coli (E. coli) that controls the biosynthesis of the amino acid tryptophan.
  • It is a classic example of a repressible operon, meaning it is usually ON but can be turned OFF when tryptophan is abundant.

Components of Trp Operon

  1. Structural Genes (trpE, trpD, trpC, trpB, trpA)

    • Encode enzymes required for synthesis of tryptophan.

  2. Regulatory Elements

    • Promoter (P): Site where RNA polymerase binds.

    • Operator (O): Binding site for the repressor.

    • Regulator Gene (trpR): Produces an inactive repressor protein.

    • Leader Sequence (trpL): Involved in fine regulation by attenuation.

Mechanism of Regulation

  1. When Tryptophan is Absent (Operon ON):

    • Repressor protein is inactive and cannot bind the operator.

    • RNA polymerase binds to promoter and transcribes the structural genes.

    • Enzymes are synthesized → Tryptophan is produced.

  2. When Tryptophan is Present (Operon OFF):

    • Tryptophan acts as a co-repressor.

    • It binds to the inactive repressor protein, activating it.

    • The active repressor binds to the operator and blocks RNA polymerase.

    • Transcription of structural genes stops → No unnecessary tryptophan synthesis.

Attenuation (Extra Control Mechanism)

  • In addition to repression, the trp operon also uses attenuation for regulation.

  • The leader region (trpL) contains short sequences that can form hairpin loops in mRNA.

  • When tryptophan levels are high → ribosome moves quickly → forms a terminator loop → transcription stops early.

  • When tryptophan levels are low → ribosome stalls → forms anti-terminator loop → transcription continues.

 

Regulation in Eukaryotes

Gene regulation in eukaryotes is more complex than in prokaryotes because:

  • DNA is packaged into chromatin.

  • Genes are separated by introns and exons.

  • Multiple levels of control exist (before transcription to after protein synthesis).

This regulation ensures cell differentiation, development, and adaptation.

Levels of Gene Regulation in Eukaryotes

1. Epigenetic Regulation (Chromatin Level)

  • Histone Modification: Acetylation, methylation, phosphorylation of histones control accessibility of DNA.

    • Histone acetylation: Opens chromatin → increases transcription.

    • Histone deacetylation: Condenses chromatin → decreases transcription.

  • DNA Methylation: Addition of methyl groups to cytosine → silences genes.

  • Chromatin Remodeling Complexes: Rearrange nucleosomes to allow or block transcription.

2. Transcriptional Regulation

  • Promoters: DNA sequences where RNA polymerase binds.

  • Enhancers & Silencers: Regulatory sequences that increase or decrease transcription.

  • Transcription Factors: Proteins that bind DNA to regulate gene expression (activators & repressors).

  • Mediator Complex: Connects transcription factors with RNA polymerase.

3. Post-Transcriptional Regulation

  • Alternative Splicing: A single pre-mRNA can give rise to different mRNAs and proteins.

  • RNA Editing: Nucleotide modifications can change coding information.

  • RNA Transport: Export of mRNA from nucleus to cytoplasm can be controlled.

  • mRNA Stability: Poly-A tail length and binding proteins regulate how long mRNA remains functional.

4. Translational Regulation

  • Control of Initiation: Proteins and initiation factors regulate the binding of ribosomes to mRNA.

  • miRNA & siRNA: Small RNAs bind to mRNA and prevent translation or degrade it (RNA interference).

5. Post-Translational Regulation

  • Protein Folding & Modifications: Proteins may undergo phosphorylation, glycosylation, acetylation.

  • Protein Degradation: Ubiquitin-proteasome system marks proteins for destruction.

  • Compartmentalization: Proteins activated only when transported to correct organelle.

 

Gene Dosage and Gene Amplification

1. Gene Dosage

  • Definition: Gene dosage refers to the number of copies of a particular gene present in a cell or organism.

  • Normally, each gene is present in two copies (diploid) in humans.

  • If the number of gene copies changes, the expression level of that gene also changes.

Effects of Gene Dosage:

  • Increased dosage: More copies → excess production of gene product (protein).

  • Decreased dosage: Fewer copies → reduced gene product.

Examples:

  • Down Syndrome (Trisomy 21): Extra copy of chromosome 21 → higher gene dosage.

  • Turner Syndrome (XO): Only one X chromosome → lower gene dosage of X-linked genes.

2. Gene Amplification

  • Definition: Gene amplification is the increase in the number of copies of a specific gene within a cell.

  • It is a controlled process and often occurs when cells need large amounts of a specific product.

Examples:

  • rRNA Genes: Amplified in rapidly dividing cells to meet high protein synthesis demand.

  • Oncogenes in Cancer: Amplification of genes like HER2/neu in breast cancer leads to uncontrolled cell growth.

  • In Insects: Amplification of detoxifying enzyme genes provides resistance against pesticides.

Difference Between Gene Dosage and Gene Amplification

Feature Gene Dosage Gene Amplification
Definition Change in number of whole gene copies due to chromosomal abnormalities. Increase in copies of a particular gene within the genome.
Cause Chromosome gain/loss (aneuploidy). Specific gene replication.
Effect Global effect on all genes of that chromosome. Local effect on a single/few genes.
Example Down syndrome (extra chromosome 21). HER2 gene amplification in cancer.

 

Generation of Antibody Diversity

  • The immune system can produce millions of different antibodies to recognize a vast array of antigens, even though the genome contains a limited number of antibody genes.
  • This diversity is generated by several mechanisms in B-cells.

1. V(D)J Recombination

  • Definition: Random rearrangement of gene segments in B-cells.

  • Components:

    • V (Variable) segments

    • D (Diversity) segments – only in heavy chains

    • J (Joining) segments

  • During B-cell development, different V, D, and J segments are combined to create a unique variable region of the antibody.

  • Result: Generates a large variety of antigen-binding sites.

2. Junctional Diversity

  • During V(D)J recombination, random addition or deletion of nucleotides occurs at the joining sites.

  • This further increases variability in the antibody’s antigen-binding site.

3. Somatic Hypermutation

  • Occurs after antigen stimulation in mature B-cells.

  • Introduces point mutations in the variable region of antibody genes.

  • B-cells producing higher-affinity antibodies are selected → affinity maturation.

4. Class Switch Recombination (Isotype Switching)

  • Changes the constant region of the antibody heavy chain without altering the antigen specificity.

  • Allows the antibody to switch from IgM → IgG, IgA, or IgE depending on the immune response.

5. Combinatorial Association

  • Each antibody has two heavy chains and two light chains.

  • Different combinations of heavy and light chains further increase diversity.