Gene Expression: From DNA to Functional Protein

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, and translation.

• Gene: A segment of DNA that contains instructions for producing a protein or functional RNA.

• Gene Product: The final molecule produced from a gene, which may be a protein or a noncoding RNA.

Genes and the Human Genome

Gene Structure

• Promoter: DNA sequence where transcription begins.

• Transcription Start Site: The location where RNA synthesis initiates.

• Coding Region: DNA sequence that is transcribed and translated into protein.

• Untranslated Regions (UTRs): Noncoding sequences at the 5' and 3' ends of mRNA.

• 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

• ~3.2 billion nucleotide pairs.

• ~21,000 protein-coding genes.

• ~9,000 noncoding RNA genes.

• >20,000 pseudogenes.

• Protein-coding exons: ~1.5% of genome.

• Highly conserved sequences: ~3.5%.

• Repetitive DNA: ~50%.

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 from the same genome by controlling which genes are expressed, when, and to what extent.

• Different genes are transcribed and translated at varying levels.

• Variation in transcription is a major determinant of protein abundance.

Importance of Gene Regulation

• Allows for cellular diversity and specialization.

• Determines cellular structure and function.

RNA: Structure and Function

General Features

• Usually single-stranded.

• Contains ribose sugar.

• Uses uracil (U) instead of thymine (T).

RNA Structure

• Can form complex 3D 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, snRNA).

Types of RNA

• mRNA (Messenger RNA): Carries genetic information from DNA to ribosomes for protein synthesis.

• rRNA (Ribosomal RNA): Forms the core of ribosome structure and catalyzes peptide bond formation.

• tRNA (Transfer RNA): Adaptor molecule that matches amino acids to mRNA codons during translation.

• snRNA (Small Nuclear RNA): Involved in RNA splicing and other nuclear processes.

• snoRNA (Small Nucleolar RNA): Guides chemical modification and processing of rRNA.

• lncRNA (Long Noncoding RNA): Regulates gene expression and organizes chromatin.

• miRNA (MicroRNA): Regulates gene expression by blocking translation or promoting mRNA degradation.

• siRNA (Small Interfering RNA): 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 catalyzed by RNA polymerase.

• Both DNA strands can serve as templates for different genes.

• Each gene is independently regulated.

• Transcription starts 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 primer or helicase.

• Error rate: ~1 per 1000 nucleotides.

Eukaryotic RNA Polymerases

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

RNA Synthesis

• Coding Strand: Same sequence as RNA (except T replaced by U).

• Template Strand: Complementary to RNA transcript.

• RNA synthesis proceeds 5′ → 3′.

Promoters and Terminators

• Promoters: DNA sequences marking transcription start sites.

• Terminators: DNA sequences signaling transcription termination.

• Promoter orientation determines template strand.

Consensus Sequences

• Short, conserved DNA motifs in promoters.

• Help RNA polymerase recognize transcription start sites.

Prokaryotic Transcription

• Initiation: RNA polymerase holoenzyme (with sigma factor) binds promoter, forms closed then open complex.

• Elongation: Sigma factor dissociates; RNA polymerase synthesizes RNA.

• 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

• TFIID (includes TBP, binds TATA box)

• TFIIA, TFIIB, TFIIF, TFIIE, TFIIH (unwinds DNA, activates polymerase)

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

• Addition of 7-methylguanosine cap via 5′-to-5′ triphosphate linkage.

• Functions: nuclear export, RNA stability, translation initiation.

RNA Splicing

• Exons: Coding sequences retained in mRNA.

• Introns: Noncoding sequences removed.

• Spliceosome: Complex of snRNAs (U1, U2, U4, U5, U6) and proteins (snRNPs) that removes introns.

• Forms a lariat intermediate; joins exons.

Alternative Splicing

• Allows different combinations of exons in mature mRNA.

• Increases protein diversity without increasing gene number.

• Mutations can disrupt splicing, causing disease.

Polyadenylation

• 3′ end of mRNA is cleaved at AAUAAA sequence.

• Poly-A polymerase adds 150–250 adenines (poly-A tail).

• Functions: RNA stability, export, translation.

RNA Termination and Export

• RNA polymerase disengages; RNA exosome degrades defective/excess RNAs.

• Processed mRNAs are exported to cytoplasm via nuclear pores.

Noncoding RNA and Nuclear Organization

Nucleolus

• Assembles ribosomal subunits.

• Produces noncoding RNAs.

• Coordinates rRNA processing and ribosome assembly.

Subnuclear Structures

• Cajal bodies: snRNA and snoRNA maturation.

• Interchromatin granules: Store processing components.

rRNA Processing

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

• 5S rRNA is synthesized separately.

The Genetic Code

Codons

• mRNA is read in triplets (codons); 64 possible codons.

• 61 codons specify amino acids; 3 are stop codons.

• The code is universal and redundant.

Start and Stop Signals

• Start codon: AUG (methionine).

• Stop codons: UAA, UAG, UGA.

Mutations and Reading Frames

• Reading Frame: The way nucleotides are grouped into codons.

• Frameshift Mutation: Insertions/deletions that alter the reading frame, often severely affecting protein structure.

• Types of Mutations: Transition, transversion, missense, nonsense, silent.

Transfer RNA (tRNA)

Structure and Function

• ~80 nucleotides; transcribed by RNA Polymerase III.

• Contains an anticodon and modified nucleotides.

• Adaptor between codons and amino acids.

Wobble Base Pairing

• Third codon position allows some mismatches.

• Reduces the number of tRNAs needed.

Aminoacyl-tRNA Synthetases

• One enzyme per amino acid; attaches correct amino acid to tRNA.

• Requires ATP.

Ribosomes and Translation

Ribosome Structure

• Composed of proteins and rRNAs; function as ribozymes.

Type	Subunits
Prokaryotic (70S)	50S (large), 30S (small)
Eukaryotic (80S)	60S (large), 40S (small)

• A site: Entry for aminoacyl-tRNA.

• P site: Holds tRNA with growing peptide chain.

• E site: Exit site for uncharged tRNA.

Translation Steps

• Initiation: Small subunit binds mRNA; initiator tRNA recognizes AUG; large subunit joins.

• Elongation: Charged tRNAs enter A site; peptide bonds form; ribosome translocates; elongation factors use GTP.

• Termination: Release factors bind stop codons; polypeptide is released; ribosome dissociates.

Polyribosomes

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

Protein Folding and Quality Control

Co-Translational Folding

• Proteins begin folding as they are synthesized.

• Secondary and tertiary structures form during translation.

Chaperones and Quality Control

• Molecular chaperones assist proper folding and prevent aggregation.

• Misfolded proteins are degraded by proteasomes to prevent toxic aggregates.

Antibiotics and Gene Expression

General Principle

• Antibiotics target processes unique to pathogens, minimizing host toxicity.

Targets and Examples

Antibiotic	Target/Effect
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 Events
Transcription	DNA → RNA (by RNA polymerase)
RNA Processing	5′ capping, splicing, polyadenylation
RNA Export	mRNA transported to cytoplasm
Translation	mRNA → Protein (by ribosome)
Protein Folding	Chaperones assist folding; quality control
Regulation	Gene expression modulated at each step

Additional info: This guide integrates foundational concepts and terminology for gene expression, including regulatory mechanisms, RNA processing, translation, and the impact of antibiotics. For further study, review the provided study questions to reinforce understanding of each topic.