L02 Genetics Molecular biology 1

Overview

This lecture introduces gene expression as the link between DNA sequence and disease, then works down through gene structure: how a gene is built (promoter, UTRs, exons/introns), how it is transcribed and (for protein-coding genes) translated, and what lies beyond the gene itself, the enhancers, silencers, insulators, locus control regions and transposable elements that tune when, where and how much a gene is expressed. The throughline is that abnormal gene expression, whether from the coding sequence or from these regulatory elements, is the mechanism behind many genetic disorders.

Gene expression: concept and process

• Gene expression is defined as the process by which information from a gene is used to synthesise a functional gene product, either a protein or a non-coding RNA (ncRNA). There are roughly 20,000 protein-coding genes and at least as many ncRNA genes.
• Disease is rarely “totally genetic” or “totally environmental”: most conditions sit somewhere on a spectrum between the two (examples given ranged from cystic fibrosis and Duchenne muscular dystrophy at the genetic end to motor vehicle accidents at the environmental end). Estimated contributors to health: lifestyle 53%, environment 21%, genetics/body makeup 16%, medical care 10%.
• Transcription initiation (protein-coding genes):
  1. Transcription factors bind the promoter, including the TATA box.
  2. RNA polymerase II binds, forming a transcriptional initiation complex with the transcription factors.
  3. The two DNA strands separate and RNA pol II begins mRNA synthesis directly, without needing a primer.
• Flow of information: DNA (gene) -> transcription -> mRNA -> translation -> protein. For non-coding genes: DNA -> transcription -> functional ncRNA (no translation step).
• The proportion of DNA that does not code for protein rises with organismal complexity: from about 25% in prokaryotes up to around 100% in humans (only ~1-1.5% of human DNA is protein-coding). This non-coding fraction was once dismissed as “junk” but may help explain complexity.

Gene products and their functions

• Protein functions and examples: structural (collagen), regulatory (insulin), contractile (actin, myosin), transport (haemoglobin), storage (egg white/albumin), protective (antibodies, e.g. IgG), catalytic (amylase, RNA polymerase), toxic (botulinum toxin, diphtheria toxin).
• ncRNA functions and examples: tRNA (adaptor between mRNA and protein), rRNA (catalytic role in the ribosome), eRNA and PAR (both promote transcription), miRNA and siRNA (both inhibit gene expression), lncRNA (regulates gene expression), snoRNA (chemical modification of RNAs), piRNA (post-transcriptional silencing of non-gene genetic elements).

Normal vs abnormal gene expression

• Normal gene expression means a functional gene product is made in the right amount (correct dosage), at the right time, and in the right cell type.
• Abnormal gene expression covers: too little or too much product, no product, decreased functionality, abnormal function, expression in the wrong cell type, or expression at the wrong time.
• Abnormal gene expression underlies common genetic disorders, including cystic fibrosis, Huntington’s disease, Angelman syndrome, Down syndrome, Fragile X syndrome, Tay-Sachs disease, Duchenne muscular dystrophy, and thalassemia.

Gene and DNA structure

• A gene is a sequence of DNA that specifies production of a functional product.
• DNA building blocks: a nucleotide = base + deoxyribose sugar + phosphate. Bases split into pyrimidines (cytosine, thymine) and purines (adenine, guanine). Base pairing: T-A via two hydrogen bonds, C-G via three hydrogen bonds. Strands run antiparallel (5’ to 3’ in opposite directions), linked by a sugar-phosphate backbone (3’ carbon of one sugar to 5’ carbon of the next).
• Genome organisation: every nucleated cell carries its own full copy of the genome. About 99% of human DNA is arranged into 46 chromosomes (23 pairs: 22 autosome pairs plus 1 sex chromosome pair, one maternal and one paternal copy of each). Maternal and paternal copies of a gene are usually not 100% identical in sequence, i.e. they are alleles. About 1% of human DNA is mitochondrial (37 genes), which is maternally inherited only.
• Cell ploidy: somatic (body) cells are diploid (2n); gametes (sex/reproductive cells) are haploid (n).

Eukaryotic gene structure and splicing

• Linear structure of a gene: additional regulatory sequences, promoter (containing the TATA box, an AT-rich region around position -25), transcriptional start (+1), 5’ UTR, coding sequence, 3’ UTR, polyadenylation signal, transcriptional stop. The coding sequence itself has a translational start (ATG) and stop (TAG/TGA/TAA).
• After transcription, the mRNA carries a 5’ G cap (m7G, used for ribosome binding), 5’ UTR, coding sequence (with AUG start and UAG/UGA/UAA stop), 3’ UTR, and a poly-A tail.
• The coding sequence is not continuous: it is split into exons interrupted by introns (e.g. exon 1, intron 1, exon 2, intron 2…). Transcription first produces pre-mRNA, which contains both exons and introns (with the 5’ cap and poly-A tail already added). Splicing then removes the introns, leaving mature mRNA made only of the joined exons, which is then translated into protein.
• The adult beta-globin gene was used as a worked example of this structure: exons correspond to the mature mRNA sequence, introns and flanking sequence are non-coding, the CAT and TATA boxes sit in the 5’ flanking region, GT/AG dinucleotides mark the intron-exon splice junctions, and an AATAAA signal marks the site for poly-A addition; the ATG initiator and TAA stop codon flank the coding sequence.

The annotated beta-globin sequence figure (source pages 29-30) is dense: only the labelled structural features (exon/intron boundaries, start codon, splice sites) could be read at the transcribed resolution; the full base-by-base sequence was not independently verified against the image.

Regulatory sequences beyond the gene

• Beyond the promoter/UTR/exon-intron structure, further elements can modulate expression: enhancers and silencers, insulators, and locus control regions (LCRs).
• Enhancers: short (50-1500 bp) DNA regions that can sit far from the gene they regulate (thousands of bp away), upstream, downstream, or within it, and work independent of their position and orientation relative to the transcriptional start site (unlike promoters). They bind transcription factors that act as activators, increasing the rate of transcription. DNA looping brings enhancer-bound activators into contact with the promoter, allowing RNA polymerase II to bind and transcription to proceed.
• Silencers: same features and mechanism as enhancers, but bind transcription factors that act as inhibitors, decreasing (or abolishing) transcription via the same DNA-looping mechanism.
• Transposable elements (“jumping genes”): DNA sequences that can move within the genome, making up about 45% of the human genome. They are considered “selfish” genes with no apparent benefit to the host cell. They can cause genetic disease if they jump into a protein-coding gene, an ncRNA gene, or a regulatory element. Many carry their own promoter, so a TE that lands near a gene can drive abnormal expression of that gene. Two mechanisms of transposition: cut-and-paste (the element is excised from its original site and inserted at a new target site) and copy-and-paste (a copy is inserted at a new site while the original remains).
• Take-home point from the lecture’s own summary: several types of DNA sequence located far from the coding sequence they influence can significantly increase or decrease that gene’s expression; the individual details and examples are secondary to understanding this general principle.

Self-test

1. Define gene expression, including the two categories of gene product it can produce.
2. Describe the three steps of transcription initiation for a protein-coding gene.
3. Distinguish normal from abnormal gene expression, and give two ways expression can be abnormal.
4. List four categories of protein function given in the lecture, each with one example.
5. List three ncRNA types described as inhibiting gene expression or as functioning within the ribosome/spliceosome-related machinery, and state each one’s role.
6. Describe the base-pairing rules in DNA, including the number of hydrogen bonds for each pair.
7. Explain what is meant by DNA strands being “antiparallel”.
8. Describe the human genome’s chromosome organisation, including the autosome/sex chromosome split and the origin of mitochondrial DNA.
9. Distinguish somatic cells from gametes in terms of ploidy.
10. Describe the structure of a eukaryotic gene from promoter to transcriptional stop, and explain how pre-mRNA differs from mature mRNA.
11. Distinguish enhancers from silencers in terms of mechanism and effect.
12. Explain why a transposable element landing near a gene can cause abnormal expression of that gene, and name the two mechanisms of transposition.
13. A gene’s coding sequence is intact, but a mutation destroys a distant enhancer that normally activates it in liver cells. Predict the effect on that gene’s expression in the liver, and explain why this would not show up as a coding-sequence mutation on gene sequencing.

Answers

Reveal answers

1. Gene expression is the process by which information from a gene is used to synthesise a functional gene product; that product is either a protein or a non-coding RNA (ncRNA).
2. (1) Transcription factors bind the promoter, including the TATA box; (2) RNA polymerase II binds, forming a transcriptional initiation complex with the transcription factors; (3) the two DNA strands separate and RNA pol II begins mRNA synthesis without needing a primer.
3. Normal expression produces a functional gene product in the right amount, at the right time, and in the right cell type. Abnormal expression is any deviation from this, e.g. too little or too much product, no product at all, decreased or abnormal function of the product, or expression in the wrong cell type or at the wrong time.
4. Structural (collagen), regulatory (insulin), transport (haemoglobin), catalytic (amylase or RNA polymerase). [Also acceptable: contractile (actin/myosin), storage (albumin), protective (antibodies), toxic (botulinum/diphtheria toxin).]
5. miRNA and siRNA both regulate (inhibit) gene expression; rRNA has a catalytic function in the ribosome.
6. Thymine pairs with adenine via two hydrogen bonds; cytosine pairs with guanine via three hydrogen bonds.
7. The two DNA strands run in opposite 5’ to 3’ directions relative to each other, rather than both running the same way.
8. About 99% of human DNA is arranged into 46 chromosomes as 23 pairs (one maternal, one paternal copy of each): 22 autosome pairs plus 1 sex chromosome pair. About 1% of DNA is mitochondrial (37 genes), inherited only from the mother.
9. Somatic cells are diploid (2n); gametes are haploid (n).
10. A gene runs promoter (with TATA box) -> transcriptional start -> 5’ UTR -> coding sequence (exons and introns) -> 3’ UTR -> polyadenylation signal -> transcriptional stop. Pre-mRNA is the initial transcript and still contains introns; splicing removes the introns to leave mature mRNA made only of joined exons, with a 5’ cap and poly-A tail, ready for translation.
11. Enhancers bind activator transcription factors and increase the rate of transcription; silencers have the same structural features and looping mechanism but bind inhibitor transcription factors and decrease transcription.
12. Many transposable elements carry their own promoter; if one inserts near a gene, its promoter can drive abnormal (e.g. ectopic or excessive) expression of that gene, or disrupt the gene/regulatory element it lands in. The two mechanisms are cut-and-paste (excision and reinsertion elsewhere) and copy-and-paste (a copy is inserted elsewhere while the original stays in place).
13. Expression of the gene in liver cells would fall (or be lost), because the enhancer is needed to bring activators into contact with the promoter via DNA looping to drive transcription in that cell type. This would not appear as a coding-sequence mutation because the enhancer lies outside the gene’s coding sequence, often thousands of base pairs away, so standard sequencing of the gene itself would look normal.