Unit 6: Genetic material and molecular mechanisms of gene expression

I. Molecular Basis of Heredity

Genetic information is encoded mainly in deoxyribonucleic acid (DNA), transferred through replication, and expressed through ribonucleic acid (RNA) and proteins. This framework developed from the identification of DNA as the transforming principle by Avery, MacLeod, and McCarty (1944), confirmation by Hershey and Chase (1952), and the double-helical model proposed by Watson and Crick using Franklin’s and Wilkins’s evidence (1953).

• Information storage: The nucleotide sequence of DNA stores hereditary instructions in a chemically stable form.
• Information transmission: Semiconservative replication copies DNA before cell division so that daughter cells inherit substantially equivalent genomes.
• Information expression: The central dogma describes the usual directional flow:
  
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    DNA --transcription--> RNA --translation--> protein

• Complementarity: Adenine pairs with thymine in DNA, or uracil in RNA; guanine pairs with cytosine.
• Sequence specificity: Different nucleotide arrangements encode different RNAs and proteins.
• Exceptions and extensions: Reverse transcriptase produces DNA from an RNA template in retroviruses, while many functional RNAs are not translated.
• Regulated expression: Cells control when, where, and how strongly genes are expressed without normally changing their DNA sequence.

II. Nucleic Acids — Forms, Organization, and Inheritance

A. Types of DNA and RNA

DNA and RNA occur in several structural and functional forms suited to information storage, transfer, regulation, and catalysis.

• DNA conformations:
  • B-DNA: The common physiological form; a right-handed helix with about 10.5 base pairs per turn and a diameter near 2 nm.
  • A-DNA: A shorter, wider right-handed helix favored by dehydrated DNA and DNA–RNA hybrids.
  • Z-DNA: A left-handed helix with a zigzag sugar-phosphate backbone, favored by some alternating purine–pyrimidine sequences.
• DNA organization: Nuclear chromosomes of eukaryotes are linear and double-stranded, whereas most bacterial chromosomes and many plasmids are circular and double-stranded.
• Unusual DNA genomes: Some viruses contain single-stranded DNA; mitochondrial and chloroplast genomes are generally circular double-stranded DNA.
• Messenger RNA (mRNA): Carries coding information from DNA to ribosomes as codons; eukaryotic mRNA commonly has a 5′ cap and 3′ poly(A) tail.
• Transfer RNA (tRNA): Acts as an adaptor by pairing its anticodon with an mRNA codon while carrying a specific amino acid at its 3′-CCA end.
• Ribosomal RNA (rRNA): Provides structural and catalytic components of ribosomes; bacterial ribosomes contain 16S, 23S, and 5S rRNAs.
• Regulatory and processing RNAs: miRNA and siRNA guide gene silencing, snRNA participates in pre-mRNA splicing, and snoRNA assists rRNA modification.
• Catalytic and genomic RNA: Ribozymes catalyze reactions, while many viruses use single- or double-stranded RNA as their genetic material.

B. Nature, structure, and replication of genetic material

Genetic material must store information, reproduce accurately, remain sufficiently stable, and permit mutation and expression.

• Chemical nature: A nucleotide contains a nitrogenous base, a pentose sugar, and phosphate; nucleotides are joined by 3′–5′ phosphodiester bonds.
• DNA polarity: Each strand has chemically distinct 5′ and 3′ ends, and paired strands run antiparallel.
• Double-helix structure: Bases face inward and pair through hydrogen bonds: A–T has two bonds, while G–C has three.
  
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    A = T
    G ≡ C

• Chargaff’s relationship: In double-stranded DNA, the molar proportions satisfy A = T and G = C, so total purines equal total pyrimidines.
• Genome packaging: Eukaryotic DNA wraps around histone octamers to form nucleosomes; approximately 147 base pairs surround each histone core.
• Semiconservative replication: Each daughter duplex contains one parental strand and one newly synthesized strand, as demonstrated by Meselson and Stahl (1958).
• Initiation: Replication begins at origins; helicase separates strands, single-strand-binding proteins stabilize them, and topoisomerase relieves torsional strain.
• Polymerization: Primase supplies an RNA primer, and DNA polymerase adds deoxyribonucleotides only to a 3′-OH group:
  
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    (DNA)n + dNTP → (DNA)n+1 + PPi

  
  Here, n is the initial chain length, dNTP is an incoming deoxyribonucleoside triphosphate, and PPi is pyrophosphate.
• Strand asymmetry: The leading strand is synthesized continuously, whereas the lagging strand forms discontinuous Okazaki fragments subsequently joined by DNA ligase.
• Accuracy: Complementary base selection and 3′→5′ proofreading reduce errors; mismatch-repair systems correct additional post-replication defects.
• Chromosome ends: Eukaryotic telomerase extends telomeres using an internal RNA template, helping solve incomplete lagging-strand replication at linear chromosome ends.

III. Protein Expression — From Nucleotide Sequence to Polypeptide

A. Protein synthesis

Protein synthesis converts the nucleotide sequence of an expressed gene into the amino-acid sequence of a polypeptide.

• Genetic code: mRNA is read in non-overlapping nucleotide triplets called codons; 64 codons specify 20 amino acids and termination signals.
• Code properties: The code is degenerate because several codons may specify one amino acid, but unambiguous because each codon has only one meaning in a given code.
• Start and stop signals: AUG usually initiates translation and specifies methionine; UAA, UAG, and UGA are termination codons.
• Amino-acid activation: Each aminoacyl-tRNA synthetase links an amino acid to the correct tRNA using ATP, producing aminoacyl-tRNA.
• Ribosomal sites: The A site receives aminoacyl-tRNA, the P site holds peptidyl-tRNA, and the E site releases uncharged tRNA.
• Peptide formation: Ribosomal rRNA catalyzes peptide-bond formation, demonstrating that the ribosome functions as a ribozyme.
• Directionality: Ribosomes read mRNA 5′→3′, while the polypeptide grows from its amino terminus toward its carboxyl terminus.
• Post-translational processing: Folding, proteolytic cleavage, phosphorylation, glycosylation, and intracellular targeting convert many new polypeptides into functional proteins.

B. Transcriptional and translational mechanisms of genetic material

Gene expression proceeds through mechanistically distinct transcriptional and translational stages.

1. Transcription:

   • Initiation: RNA polymerase binds a promoter; bacterial sigma factors and eukaryotic general transcription factors help recognize promoter sequences.
   • Elongation: RNA polymerase reads the DNA template strand 3′→5′ and synthesizes complementary RNA 5′→3′ without requiring a primer.
   • Termination: Specific signals release the RNA transcript and polymerase; bacterial termination may be intrinsic or rho-dependent.
   • Eukaryotic processing: Pre-mRNA receives a 5′ cap, undergoes intron removal by the spliceosome, and gains a 3′ poly(A) tail.
   • Template relationship: The RNA sequence matches the DNA coding strand except that uracil replaces thymine.

2. Translation:

   • Initiation: The small ribosomal subunit positions the initiator tRNA at the start codon; the large subunit then completes the initiation complex.
   • Elongation: Aminoacyl-tRNAs enter the A site, peptide bonds form, and the ribosome translocates by one codon using GTP-dependent factors.
   • Termination: A release factor recognizes a stop codon and promotes hydrolysis of the completed polypeptide from its tRNA.
   • Polysomes: Multiple ribosomes can translate one mRNA simultaneously, increasing protein production.
   • Prokaryotic coupling: Because bacteria lack a nucleus, translation may begin on an mRNA while it is still being transcribed; eukaryotic transcription and translation are spatially separated.

IV. The Gene — Definition, Organization, Activity, and Control

A. Gene concept

A gene is a DNA sequence that contributes to a functional RNA or polypeptide, together with sequences required for its proper expression.

• Classical concept: Mendel’s hereditary “factors” were inferred from predictable segregation and independent assortment of traits.
• Chromosomal concept: Morgan’s work connected genes with specific chromosome positions called loci.
• One gene–one enzyme concept: Beadle and Tatum linked genes to biochemical reactions; the idea was refined to one gene–one polypeptide.
• Functional concept: A gene may encode a protein or a functional RNA such as rRNA, tRNA, or miRNA.
• Complexity: Alternative splicing allows one eukaryotic gene to produce several RNA and protein isoforms, so a gene is not always equivalent to one product.
• Cistron: A cistron is a functional genetic unit defined by complementation analysis and generally corresponds to a polypeptide-coding sequence.

B. Gene structure

Gene structure comprises transcribed regions and regulatory sequences that determine the identity and abundance of the gene product.

• Prokaryotic genes: A promoter is followed by a transcribed region and terminator; several coding regions may form one polycistronic operon.
• Eukaryotic promoters: Core promoter elements recruit RNA polymerase II and general transcription factors; some promoters contain a TATA box.
• Transcription unit: It includes a 5′ untranslated region, coding sequence, 3′ untranslated region, and RNA-processing signals.
• Exons and introns: Exons remain in mature RNA, whereas introns are removed from pre-mRNA by splicing.
• Regulatory elements: Enhancers and silencers may act over long distances through DNA looping and transcription-factor binding.
• Reading frame: Translation depends on the start site because shifting the nucleotide grouping changes every downstream codon.

C. Gene function

Genes function by producing molecules that establish cellular structures, catalyze reactions, transmit signals, and control other genes.

• Structural products: Genes encode proteins such as collagen, actin, and membrane channels.
• Enzymatic products: Enzyme-coding genes influence metabolic pathways; loss of phenylalanine hydroxylase activity causes phenylketonuria.
• Regulatory products: Transcription factors bind specific DNA sequences, while regulatory RNAs alter mRNA stability or translation.
• Genotype-to-phenotype pathway: Gene products interact with other genes and environmental factors, so most phenotypes are not determined by a single sequence alone.
• Mutation effects: Substitutions, insertions, deletions, or regulatory mutations may produce loss-of-function, gain-of-function, or altered-expression alleles.
• Dosage and timing: Correct product quantity and developmental timing can be as important as the product’s amino-acid sequence.

D. Gene regulation

Gene regulation adjusts gene-product abundance in response to developmental programs, environmental conditions, and cellular signals.

• Bacterial operons: Functionally related genes share regulatory DNA and are transcribed together.
• Lac operon: Without lactose, the LacI repressor binds the operator; allolactose reduces repression, while low glucose promotes CAP–cAMP activation.
• Transcriptional control: Activators and repressors change RNA-polymerase recruitment or activity at promoters.
• Epigenetic control: DNA methylation and histone modifications influence chromatin accessibility without changing nucleotide sequence.
• RNA-level control: Alternative splicing, RNA editing, polyadenylation, localization, and regulated degradation alter mature RNA output.
• Translational control: Initiation factors, RNA-binding proteins, and miRNAs regulate ribosome recruitment and translation rate.
• Protein-level control: Proteolysis, phosphorylation, and compartmentalization modify protein concentration or activity.
• Biological significance: Differential gene expression allows cells with essentially the same genome to develop into specialized cell types and respond rapidly to changing conditions.