Eukaryotic Genome Structure Flashcards
C Value Paradox
Discrepancies between number of genes/size and complexity of genome
Amount of non-coding DNA increases dramatically with organism complexity
C Value
Haploid DNA amount in genome
C0T Analysis
Before the genome sequencing era, the genome complexity could be assessed using renaturation kinetics of single-stranded eukaryotic DNA. Renaturation is a bimolecular reaction in which the reaction rate is directly proportional to the product of the concentrations c of the two homologous strands.
In other words, the product of the initial concentration of single-stranded DNA, c0, and the time required to renature 50% of the DNA, t1/2, is inversely proportional to the rate constant k2 of the renaturation reaction.
This analysis revealed that much of this extra DNA contains repetitive nucleotide sequences. The observed reassociation kinetics reveals the presence of three types of sequences.
This c0t1/2-value (usually simply called theCot-value) is directly proportional to the complexity of the genome (defined as the number of unique sequences in the genome).
Experimental Steps of CoT analysis
- shear DNA to 400 bp
- denature DNA
- slowly cool and sample
- determine % ssDNA at time points
Plot log C0T against %ssDNA to determine the rate of reannealing : determinant of the species genome size/complexity
Repetitive DNA renatures at low C0T values and unique DNA renatures at high levesl
Eukaryotic DNA Elements
- Single copy functional genes
- Repetitive DNA
- Spacer DNA
- 2% coding DNA
Eukaryotic Simple Transcription Unit
- control regions
- cap site
- introns and exons
- polyA site
Functional Repetitive Sequences
Families of coding genes (dispersed vs tandem gene families) and pseudogenes and Non-coding functional sequences
Pseudogene
Once functional ('zombie' gene), role in regulation/transcribed into siRNA
Transcription factor binding sitesMulti-gene Families
- Groups of identical or very similar sequences.
- can be tandemly arrayed (Head-to-tail fashion)
- Examples include the tRNA genes (at ~50 sites, containing 10-100 genes), Histone genes in some species.
- Human genome approx. 280 copies of repeat unit containing 28S,5.8S, and 18S rRNA, grouped into five clusters of 50-70 repeats.
- example is B-globin protein
Dispersed Multigene Family
- genes that have become dispersed at several locations in the genome via chromosomal arrangements
- not tandemly repeated
- they may have different functions
Non-functional repetitive sequences
- 65% of the human genome comprises intergenic regions of unknown function
- some repetitive sequences (thousands of tandem repeats) are associated with heterchromatin (non-transcriptionally active)
Transposons
- repeat sequences which have increased in copy umber through transposition
- transposable elements
- transposed via a DNA or RNA intermediate
Simple Sequence DNA
- microsatellites
- repeats < 13 bp
- scattered throughout genome
- 3% of genome
Variable Number Tandem Repeats
- includes minisatellites
- repeat units up to 25 bp in length
- associated with telomeres
Retrotransposons
- resemble retroviruses but only move within a cell rather than between cells
LINEs (long interspersed nuclear elements)
- less frequent but longer
- 1 million copies
SINEs (short interspersed nuclear elements)
- highest copy number in human genomes
- 1.7 million copies
Spacer DNA
Regulatory mechanism to promote successful transcription
- non coding DNA between genes
Importance of Chromosomes
- condense genome to allow it to fit in the cell
- without this you couldn’t replicate or transcribe genes
Nuclease Protection Experiments
- used rat liver endonuclease on chromatin (cuts non shielded DNA)
- releases multiples of smallest DNA unit
- indicates presence of protein complexes on DNA
Chromatin
the material of which the chromosomes of organisms other than bacteria (i.e. eukaryotes) are composed, consisting of protein, RNA, and DNA.
Nucleosome
DNA wrapped around a nucleosome of an octamer of histones
Forms a ‘chromatosome’
Histone Wrapping
Histone proteins form a barrel shaped core octamer
- H3/H4 dimer forms, tetramer forms, interacts with H2A:H2B dimer
Octamer interacts with 146 bp of DNA
- histones contact minor groove leaving major groover available for gene regulating expression
- Histone H1 (linker histone) locks complex with DNA
Polynucleosome
- more condensed complex
- chromatin condenses by zig-zag folding
- forms a solenoid
- H1 histone stabilises structure
- histone H2A-H2B dimer and H4
- extent of compaction dependent on cell environment
Scaffold Association
- 30 mm fibre organised as looped domains.
- Protein scaffold made of Histone H1 and other proteins (Sc1 & Sc2)
- Scaffold Attachment points (AT-Rich region)
- Radial arrangement of Loops
- loops are fixed at the base, structure can generate coils and supercoils
Chromosome Formation
A single length of DNA is wrapped many times around histones, to form structures called nucleosomes. These nucleosomes then coil up tightly to create chromatin loops. The chromatin loops are then wrapped around each other to make a full chromosome
- Euchromatin: open and transcriptionally active
- Heterochromatin: condensed and less active (can be faculative or constitutive)
Chromosome Structure
- telomere
- hetero/euchromatin
- centromere
Telomeres
Important for manoeuvring of chromosomes in cell division and protection of DNA ends
- Terminal region of chromosome
- Enable the cell’s machinery to distinguish between real ends from double strand break
- made of repeated motif
RNA processing
- transcription + capping
- cleavage
- polyadenylation
- splicing
RNA Capping
The RNA Polymerase contains a C-Terminal Domain (CTD). When Phosphorylated it recruits the Capping enzyme complex. Modifies the 5’-end to a 7-methylguanosine, joined by a 5’-5’- triphosphate bridge.
- Guanylyl transferase removes the γ- phosphate of 5’-nucleotide and β- & γ- phosphate of GTP.
- New terminal guanosine converted to 7-methylguanosine by methyl group attached to nitrogen 7 of purine ring (by guanine methyltransferase with the help of S-adenosylmethionine).
Uses of Capping
- decapping makes it accessible to nuclease
- capping regulates nuclear export, promote translation, prevent nuclease degradation, promote 5’ proximal intron excision
Polyadenylation
- PolyA polymerase add adenyl 3’ends (250 adenosines
- The Poly(A) signal & GU rich region are binding sites for the ‘Cleavage & Polyadenylation Specificity Factor’ (CPSF) and the ‘Cleavage Stimulation Factor’ (CstF)
- polyadenylation influences mRNA stability to prevent degradation
Splicing
- splices out intron sequences
- Found in the “GU-AG” Introns these sequences probably act as recognition for RNA-binding proteins
- intron consensus sequence: donor site + branchpoint A + acceptor site
Transester-fication Reaction
- In the first transesterification reaction, the ester bond between the 5′ phosphorus of the intron and the 3′ oxygen of exon 1 is exchanged for an ester bond with the 2′ oxygen of the branch-site Adenosine. Begins to form a lariat structure.
- the ester bond between the 5′ phosphorus of exon 2 and the 3′ oxygen of the intron is exchanged for an ester bond with the 3′ oxygen of exon 1
- the intron is released as a lariat structure and the two exons have been “spliced”
Spliceosome
- carry out splicing
- snRNA + protein + mRNA = snRNP
- forms the spliceosome
Alternative Splicing
Differentially spliced transcripts may lead to proteins with differing cellular destinations and different catalytic or interactive properties
Dscam gene
- neuronal adhesion of Drosophila
- final mRNA contains 24 exons, four of which are arrays of alternative exons
- 38,000 possible splicing combinations
Group 1 Introns
- essential genes
- Found in pre-rRNA
- 2 transesterifications
- 1st induced by free nucleoside/nucletoide (GTP)
- Attacks 5’ splice site
G transferred to the 5’ end - 2nd involves 3’-OH and causes cleavage
- Autocatalytic- Ribozyme!
Group 2 Introns
- noncoding regions
- exon 1 recognises 5’P of AG of exon 2
- same mechanism as spliceosome
- not ATP/GTP dependent
- cleavage at 5’ splice site
- formation of lariat like intermediate
- cleavage at 3’ spice site
- ligation of exons
Uses of Introns
Comparison between related genes in an organism or between same gene in different organisms shows that intron sequences are poorly conserved. But introns usually undergo rearrangements rather than point mutations caused by transposable elements.
- some introns so large they include complete gene
- introns may have regulatory sequences controlling expression of the gene
- most introns can be deleted without immediate major effect on the gene (no functional selection allows genome evolution)
- alternative splicing enhances coding potential
