Gene Expression: From DNA to Functional Protein

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Gene Expression

General Process

Gene expression is the process by which information encoded in a gene is used to direct the synthesis of a functional gene product, typically a protein or a functional RNA molecule. This process involves multiple steps, including transcription, RNA processing, translation, and protein folding.

Gene: A hereditary unit consisting of DNA that contains instructions for the characteristics of an organism. Genes can encode proteins or functional RNAs.
Gene Product: The final functional molecule produced from a gene, which may be a protein or a noncoding RNA.

Genes and the Human Genome

Gene Structure

Genes are organized into distinct regions that regulate and encode their function.

Promoter: DNA sequence where transcription machinery assembles.
Transcription Start Site: The location where RNA synthesis begins.
Coding Region: DNA sequence that is transcribed and translated into protein.
Untranslated Regions (UTRs): Noncoding sequences at the 5' and 3' ends of mRNA that regulate translation and stability.
Exons: Expressed sequences retained in mature mRNA.
Introns: Noncoding sequences removed during RNA splicing.
Termination Site: Sequence signaling the end of transcription.

Human Genome Composition

The human genome contains approximately 3.2 billion nucleotide pairs.
There are about 21,000 protein-coding genes and 9,000 noncoding RNA genes.
More than 20,000 pseudogenes are present.
Protein-coding exons account for only about 1.5% of the genome.
Highly conserved sequences make up ~3.5% of the genome.
Repetitive DNA constitutes about 50% of the genome.

Gene Organization

Genes are separated by intergenic regions.
Chromosomes contain both protein-coding and noncoding genes.

Gene Regulation

Differential Expression

Gene regulation ensures that different cell types can arise and function despite having identical genomes.

Genes are transcribed and translated at varying levels according to cellular needs.
Variation in transcription is a major determinant of protein abundance.
Gene regulation determines cellular structure and function.

RNA: Structure and Function

General Features

RNA is usually single-stranded.
Contains ribose sugar instead of deoxyribose.
Uses uracil (U) instead of thymine (T).

RNA Structure

Can form complex three-dimensional structures via intramolecular base pairing.
Structure is sequence-dependent and critical for function.

Functions of RNA

Information storage (e.g., mRNA).
Catalysis (e.g., rRNA, ribozymes).
Regulation (e.g., miRNA, siRNA).
Structural support (e.g., rRNA in ribosomes).

Types of RNA

Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes; codes for proteins.
Ribosomal RNA (rRNA): Forms the core of ribosomes and catalyzes peptide bond formation.
Transfer RNA (tRNA): Adaptor molecule that matches amino acids to mRNA codons during translation.
Small Nuclear RNA (snRNA): Involved in RNA splicing and other nuclear processes.
Small Nucleolar RNA (snoRNA): Guides chemical modification and processing of rRNA.
Long Noncoding RNA (lncRNA): Regulates gene expression and organizes chromatin.
MicroRNA (miRNA): Regulates gene expression by blocking translation or promoting mRNA degradation.
Small Interfering RNA (siRNA): Degrades specific mRNAs and regulates chromatin.
piRNA: Protects germ cells from transposable elements.

Transcription

Definition and General Features

Transcription is the synthesis of RNA from a DNA template. It is the first step in gene expression and is required because DNA cannot be directly translated into protein.

Both DNA strands can serve as templates for different genes.
Each gene is independently regulated.
Transcription begins and ends at specific sites.
Not all transcripts encode proteins.

RNA Polymerase

Catalyzes the addition of ribonucleotides to the growing RNA chain.
Does not require a helicase or primer.
Error rate: ~1 error per 1000 nucleotides.

Eukaryotic RNA Polymerases

RNA Polymerase I: Transcribes 5.8S, 18S, and 28S rRNA genes.
RNA Polymerase II: Transcribes protein-coding genes and many regulatory RNAs.
RNA Polymerase III: Transcribes tRNAs, 5S rRNA, and several small RNAs.

RNA Synthesis

Coding Strand: Has the same sequence as the RNA transcript (except T replaced by U).
Template Strand: Complementary to the RNA transcript.
RNA synthesis proceeds in the 5′ → 3′ direction.

Promoters and Terminators

Promoters: Mark transcription start sites and determine template strand orientation.
Terminators: Mark transcription stop sites.
Consensus Sequences: Shared sequence motifs in promoters that help RNA polymerase recognize start sites.

Prokaryotic Transcription

Initiation: RNA polymerase holoenzyme, guided by sigma factor, binds promoter and forms closed then open complex.
Elongation: Sigma factor dissociates; RNA polymerase synthesizes RNA until a terminator is reached.
Termination: Specific DNA sequences trigger release of RNA.

Eukaryotic Transcription

More complex; requires many transcription factors.
RNA Polymerase II cannot initiate transcription alone.
General transcription factors (e.g., TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH) are required.
Initiation involves TBP binding the TATA box, assembly of factors, DNA unwinding by TFIIH, and activation of RNA Polymerase II.
Regulation involves activators, enhancers, mediator complexes, chromatin remodeling, and histone modification.

RNA Processing

Overview

Eukaryotic mRNAs undergo several processing steps before translation, often while transcription is ongoing.

5′ capping
RNA splicing
3′ polyadenylation
RNA export

5′ Capping

A 7-methylguanosine cap is added to the 5′ end via a 5′-to-5′ triphosphate linkage.
Functions: Required for nuclear export, protects RNA from degradation, promotes translation initiation.

RNA Splicing

Exons: Expressed sequences retained in mRNA.
Introns: Noncoding sequences removed during splicing.
Spliceosome: Complex of snRNAs (U1, U2, U4, U5, U6) and proteins (snRNPs) that removes introns via a lariat intermediate.
Alternative Splicing: Allows different combinations of exons, increasing protein diversity.
Clinical Relevance: Mutations affecting splicing can cause disease.

Polyadenylation

The 3′ end of mRNA is cleaved at an AAUAAA sequence.
Poly-A polymerase adds ~150–250 adenines (poly-A tail).
Functions: Increases RNA stability, extends mRNA lifetime, aids export and translation.

RNA Termination and Export

RNA polymerase disengages from DNA; RNA degradation may contribute to termination.
RNA Exosome: Nuclear complex with exonucleases that removes defective/excess RNAs.
Processed mRNAs are packaged with proteins and exported through nuclear pores.

Noncoding RNA and Nuclear Organization

Nucleolus

Assembles ribosomal subunits.
Produces noncoding RNAs.
Coordinates rRNA processing and ribosome assembly.

Subnuclear Structures

Cajal Bodies: Involved in snRNA and snoRNA maturation.
Interchromatin Granules: Store RNA processing components.

rRNA Processing

45S precursor rRNA is modified and cleaved to produce 18S, 5.8S, and 28S rRNAs.
5S rRNA is synthesized separately.

Genetic Code

Codons and Properties

mRNA is read in triplets called codons (64 possible codons).
The genetic code is universal and redundant (degenerate).
61 codons specify amino acids; 3 are stop codons (UAA, UAG, UGA).
Translation begins at AUG (methionine).

Mutations and Reading Frames

Reading Frame: The way nucleotides are grouped into codons; must be correct for proper protein synthesis.
Types of Mutations:
- Transition: Purine to purine or pyrimidine to pyrimidine change.
- Transversion: Purine to pyrimidine or vice versa.
- Missense: Changes one amino acid.
- Nonsense: Introduces a stop codon.
- Silent: No change in amino acid.
Frameshift Mutations: Insertions or deletions that alter the reading frame, often severely affecting protein structure.

Transfer RNA (tRNA)

Structure and Function

~80 nucleotides long; transcribed by RNA Polymerase III.
Contains an anticodon and many modified nucleotides.
Acts as an adaptor between codons and amino acids.

Wobble Base Pairing

The third codon position (wobble position) allows some mismatches, reducing the number of tRNAs needed.

Aminoacyl-tRNA Synthetases

One enzyme per amino acid; attaches the correct amino acid to its tRNA using ATP.

Ribosomes

Structure and Function

Composed of proteins and rRNAs; function as ribozymes.
Prokaryotic Ribosomes: 70S (50S + 30S subunits).
Eukaryotic Ribosomes: 80S (60S + 40S subunits).
Functional sites: A (aminoacyl), P (peptidyl), and E (exit) sites.

Translation

Initiation

Small ribosomal subunit binds mRNA 5′ end.
Initiator tRNA recognizes AUG start codon.
Large subunit joins to form the initiation complex.

Elongation

Charged tRNAs enter the A site.
Peptidyl transferase forms peptide bonds.
Ribosome translocates one codon at a time; elongation factors use GTP.

Termination

Release factors bind stop codons; water is added to release the polypeptide.
Ribosome dissociates.

Polyribosomes

Multiple ribosomes can translate a single mRNA simultaneously, increasing protein production efficiency.

Protein Folding and Quality Control

Co-Translational Folding

Proteins begin folding into secondary and tertiary structures during translation.

Chaperones

Assist in proper folding, recognize exposed hydrophobic regions, and prevent aggregation.

Protein Quality Control

Misfolded proteins are degraded by proteasomes, preventing toxic aggregates.

Antibiotics and Gene Expression

General Principle

Antibiotics target processes that differ between host and pathogen cells.

Targets and Examples

Peptidoglycan synthesis, transcription, and translation are common targets.
Tetracycline: Blocks aminoacyl-tRNA binding.
Streptomycin: Disrupts translation initiation.
Chloramphenicol: Inhibits peptidyl transferase.
Erythromycin: Blocks peptide elongation.
Rifamycin: Inhibits bacterial RNA polymerase.
α-Amanitin: Inhibits eukaryotic RNA Polymerase II.

Summary Table: Key Steps in Gene Expression

Step	Main Event	Key Molecules
Transcription	DNA to RNA synthesis	RNA polymerase, transcription factors
RNA Processing	5′ capping, splicing, polyadenylation	snRNAs, spliceosome, poly-A polymerase
RNA Export	mRNA transport to cytoplasm	Export receptors, nuclear pores
Translation	mRNA to protein synthesis	Ribosomes, tRNA, elongation factors
Protein Folding	Folding into functional structure	Chaperones, proteasomes

Key Equations and Concepts

Direction of RNA Synthesis:
Genetic Code:
ATP Requirement for Aminoacyl-tRNA Synthetase:

Example

Example of Alternative Splicing: The DSCAM gene in Drosophila can generate over 38,000 different mRNAs through alternative splicing, greatly expanding protein diversity from a single gene.

Additional info: Many disease-causing mutations affect splicing, leading to genetic disorders such as spinal muscular atrophy and certain cancers.