What is DNA?
- DNA is Deoxyribonucleic Acid.
- It is a macromolecule consisting of a linear strand of nucleotides.
- Single linear strands binds to complementary strands to form double-stranded DNA.
Single Stranded DNA
5’ (5 prime) and 3’ (3 prime) are numbered based on the carbon atoms of the sugar. The base is attached to the first carbon, the phosphate links between the 3’ of one sugar and the 5’ of the adjacent sugar. Numbering starts at the carbon closest to the base.
By convention single-stranded DNA sequence is listed 5’ to 3’.
How does DNA exist in 3D?
- There are two antiparallel strands of DNA
- The bases are ‘stacked’
- There are two grooves: major and minor
Genome variation (interesting facts tbh)
- Human genome is 3x10’9 base pairs.
- It contains 20,000 genes.
- There is trend for simpler organisms to have fewer genes e.g. - flies (10,000), yeast (4,000), bacteria (1,000)
There is 2m of DNA in a nucleated cell, there are 37.2 trillion cells in your body so that’s 7.44x10’13 metres of DNA. = 250 journeys to the sun and back.
How do we pack so much DNA into cells?
They’re packed into HISTONES.
- Basic (positively charged) proteins that bind DNA.
- Eight histones 2x(H2A+H2B+H3+H4) form the nucleosome.
- Histone 1 binds the linker DNA.
What is the order in which DNA is packed?
- DNA basic helix
- nucleosomes
- chromatin fibres
- extended section of chromosome
- loops of chromatin fibre
- chromosome
What are the 3 chromosome structures?
Metacentric - long and short arm of equal length.
Submetacentric - a long long arm and short short arm
Acrocentric - Long long arm and no short arm.
Centromere - gives orientation point.
Telomeres - ends of chromosome.
Human karyotype - an individual’s collection of chromosomes.
What is the relation between DNA and genes?
Genome = The primary DNA sequence encodes all the gene products necessary for a human.
The primary DNA sequence also includes a large number of regulatory signals. Much of the DNA sequence does not have an assigned function as yet.
What is the exome?
The exome is made up of gene sequences. The exome is the part of the genome composed of exons, the sequences which, when transcribed, remain within the mature RNA after introns are removed by RNA splicing and contribute to the final protein product encoded by that gene.
Some definitions use all of the coding sequences (1.2% of genome)
Some definitions use all of the gene sequences (2% of genome)
What is a gene?
All of the DNA that is transcribed into RNA plus all of the cis-linked (local) control regions that are required to ensure quantitatively appropriate tissue-specific expression of the final protein. Promoter function - on/off, where, when, how much?
It is NOT just the bits that encode the final protein, regulation of the gene is very important.
What are the intergenic regions (in the chromosome)?
Intergenic regions contain sequences of no known function, such as repetitive DNA, endogenous retroviruses, pseudogenes. They may contain many regulatory elements.
Intergenic regions are 98% of the genome.
How is the genome organised?
Genes often cluster in families - e.g. globin clusters.
This:
- allows for coordinate gene regulation
- may just reflect evolutionary history
What do genes consist of?
Genes consist of a promoter and a transcription unit. The transcription unit consists of extrons (there are n number of extrons) and introns (n-1 introns). Some exons have coding and non-coding regions. The promoter is at the 5’ prime end. There are transcription and translation initiators and terminators.
Introns in genes
- Vary in number – from 0 to at least 311
- Vary in size - 30bp to 1Mbp
- Some introns contain other genes
- Introns are crucial because the protein repertoire or variety is greatly enhanced by alternative splicing in which introns take partly important roles. Alternative splicing is a controlled molecular mechanism producing multiple variant proteins from a single gene in a eukaryotic cell.
What are TATA boxes and regulatory elements?
TATA boxes are needed to recruit general transcription factors and RNA polymerase.
Regulatory elements are needed to regulate recruitment of RNA polymerase.
What does the promoter do?
- Promoters recruit RNA polymerase to a DNA template.
- RNA polymerase binds asymmetrically and can only move 5’ to 3’.
- Regulation occurs via transcription factors.
What are some other Regulatory Regions?
Enhancers upregulate gene expression – they are short sequences that can be in the gene or many kilobases distant. They are targets for transcription factors (activators).
Silencers downregulate gene expression. They are also position-independent and are also targets for transcription factors (repressors).
Insulators are short sequences that act to prevent enhancers/silencers influencing other genes.
What happens to mRNA in transcription with RNA polymerase II?
- Messenger RNA synthesis (transcription) is catalysed by RNA Polymerase II
- Transcribes in 5’ to 3’ direction
- Transcribes everything after the transcription start site (exons and introns)
- mRNA is post-transcriptionally modified
RNA polymerase II recognises promoters efficiently with the assistance of many other transcripton factors.
Very briefly, describe the steps of transcription.
1) RNA Polymerase recruited (closed complex)
2) DNA helix locally unwound (open compex)
3) RNA synthesis begins
4) Elongation
5) Termination
6) RNA Polymerase dissociates
Transcription –> primary transcript (exons and introns) a.k.a pre-mRNA. —> Post-transcriptional modifications ——> forms mature mRNA.
What are the post-transcriptional modifications of mRNA?
- Capping - capped at 5’ end.
Splicing - spliced introns removed.
Polyadenylations - polyadenylated at 3’ end, adding lots of As at the end.
What is capping (5’cap)?
After 25-30 nucleotides are synthesised, a methylated cap is added to the 5’ end by three enzyme activities:
- RNA 5’-triphosphatase
- Guanylyltransferase
- N7G-methyltransferase
This is added to prevent digestion.
The first two activities are carried out by a bifunctional capping enzyme (CE).
RNA Polymerase II is also required.
What is the process of splicing introns?
Splicing is removal of introns by spliceosome and by joining of exons. 150 proteins form the mature spliceosome.
Linkage 2’-5’ in the intron. The catalysing of the extrons splices the introns out, giving a ‘lariat’ structure.
Once spliced, proteins associated with the Exon Junction Complex
help recruit the Transcription Export Complex (TREX complex) allowing for the targeting mRNA for nuclear export in endoplasmic reticulum.
What happens in polyadenylation?
There is the addition of the 3’ POLY A tail.
- CPSF (Cleavage and Polyadenylation Stimulating Factor) recognises the PAS (Polyadenylation signal) and acts on cleavage site
- CSTF (Cleavage Stimulating Factor) recognises GU-rich Downstream Elements (DSE)
- PAP (Poly-A polymerase) is recruited and adds multiple A bases after cleavage site
- PAB is Poly-A Binding Protein. Other proteins appear to be required for this process – CFIm (Cleavage Factor Im), CFIIm and Simplekin
What is alternative splicing?
Exons can be skipped or added, so variations of a protein (called isoforms) can be produced from the same gene.
