oceg (pt 1) - structure and organisation of the eukaryotic genome

genome

entire genetic material of an organism

• all the DNA in a cell → coding AND non-coding sequences

• in most multicellular eukaryotes

  • only a small proportion of all the DNA in a cell consists of genes

  • most of the DNA is made up of non-coding sequences

why is there a need to pack DNA?

• there is a need for multilevel DNA packing because:

  1. a eukaryotic DNA molecule is very long → chromosomes are linear and long

  2. to compact and fit all DNA molecules into the nucleus

  3. to prevent breakage of DNA

how is DNA packaged

• eukaryotic DNA is combined with a large number of proteins to form chromatin fibres in a non-dividing cell

• the chromatin goes through multiple levels of packaging, resulting in condensed metaphase chromosomes in cells undergoing cell division → metaphase stage of mitosis and meiosis

overview of chromatin packing

• the level of DNA packing depends on whether the cell is undergoing cell division:

  1. in both interphase and dividing cells

     • DNA wraps around proteins called histones to form nucleosomes → the chain of nucleosomes coils to form the 30nm chromatin fibre → forms looped domains attached to a protein scaffold, forming the 300nm chromatin fibre

  2. in dividing cells only

     • the looped domains coil and fold further to give rise to highly condensed chromosomes

histones

• are the major structural proteins of chromosomes

• have a high proportion of positively charged amino acids (lysine and arginine)

• bind tightly to negatively charged (phosphate groups) DNA by ionic attractions

nucleosome

• nucleosome is the basic unit of DNA packing

• each nucleosome consists of DNA wound twice around 8 histone proteins

  • 4 types of molecules, 2 each

• between nucleosomes is the linker DNA → connects one nucleosome to another

• this forms the 10nm chromatin fibre

30 nm chromatin fibre

• the nucleosomes and linker DNA coil to form the 30nm chromatin fibre/solenoid

• about 6 nucleosomes are assembled in one turn in a solenoid

• histone proteins of nucleosomes, linker DNA and H1 histone interact to stabilise the 30nm chromatin fibre

  • H1 histone attaches to DNA near nucleosome

looped domains

• the 30nm fibre form looped domains

• are attached to protein scaffold (non-histone proteins), resulting in a 300 nm chromatin fibre

metaphase chromosome

only in dividing cells

• the looped domains attached to protein scaffold further coil and fold

• this compacts 300nm chromatin to form the characteristic metaphase chromosome

interphase chromatin

• less condensed than metaphase chromosome

• there are two forms:

  1. heterochromatin: highly condensed DNA (darkly stained region)

  2. euchromatin: less condensed DNA → contains genes that are undergoing transcription actively

features of a typical eukaryotic gene

• a unit of inheritance

• contains the nucleotide sequence for synthesis of a functional gene product

• includes both coding sequences (exons) and non-coding sequences (introns)

  • eukaryotic genome: 3% coding sequences and 97% non-coding sequences

• if gene product is a polypeptide chain, the sequence of nucleotides in the DNA codes for sequence of amino acids in a polypeptide chain (primary structure)

coding sequences

exons:

• sequence of nucleotides in the exon codes for sequence of amino acids (primary structure) in a polypeptide chain or code for RNA

• exons must be joined together to from a continuous coding sequence

non-coding sequences

• sequence of nucleotides that do not code for sequence of amino acids or RNA

  1. introns

  2. control elements → non-coding regulatory sequences

     • promoters

     • distal control elements → enhancers and silencers

     • terminator sequence → transcription

     • centromeres and telomeres

• the direction in which transcription takes place from the transcription start site is referred as downstream, and the opposite direction is referred to as upstream

centromeres → structure

• the centromere is a constricted region of a chromosome

• each sister chromatid has its own centromere DNA sequence

• it is composed of highly repeated, non-coding DNA sequences

• bound to kinetochore (protein) ⇒ site of attachment of spindle fibres

centromeres → functions

• joins two sister chromatids of a chromosome together

• the centromeres are the attachment sites for kinetochore and spindle fibres attach during metaphase

• during anaphase of mitosis and anaphase II of meiosis, centromeres divide and shortening of spindle fibres pull chromatids to opposite poles of the cell

telomere → structure

• DNA at both ends of each chromosome → open ends of linear chromosome

• the chromatin at the telomeres are highly condensed (heterochromatin)

• there are no genes → telomere consists of multiple short tandem repeats of non-coding nucleotide sequence

  • tandem repeat sequence in human is 5’ -TTAGGG- 3’

  • the exact repeated sequence is species specific

  • 100-10000 repeats of 5-10 nucleotides

• telomere-binding proteins bind to the ends of telomers to form telomere caps

  • these proteins enable the 3’ protruding ends to loop back and tuck the single-stranded end into the DNA, forming a t-loop

• the 3’ end at each telomere is always slightly longer than the 5’ end with which it is paired, leaving a protruding single-stranded end

telomere → function

1. telomeres prevent loss of genes near the ends of DNA with each round of DNA replication

   • DNA molecules shorten after each round of DNA replication due to end replication problem

   • telomeres are shortened without harmful effects

   • without telomeres, genes near the ends of the DNA molecule will be lost

2. telomeres bind to telomeric proteins (telomere caps) which

   • prevent ends of chromosomes from attaching to each other (complementary base pairing) and

     • chromosomes joined end to end will trigger cell apoptosis

   • from degradation by exonuclease [digests nucleic acid]

3. length of telomeres determines life span of cells

   • when telomeres reach a shortened critical length, they are detected by the DNA repair system which triggers apoptosis → cell death

4. telomeres provide the recognition site for telomerase to recognise and bind to lengthen DNA in zygotic and embryonic stem cells

telomerase

• an enzyme that catalyses lengthening of telomeres → telomerase enzyme binds to the recognition site on telomeres to lengthen telomeres

• found in zygotic stem cells, embryonic stem cells but absent/very low levels in adult stem cells( where the telomerase gene is switched off)

• it is also found in cancer cells → telomerase gene is switched on

• zygotic stem cells and embryonic stem cells divide rapidly, and DNA recognition occurs frequently, thus without telomerase, the DNA will shorten very quickly

• these cells do not undergo pre-mature cell death and keep dividing

how does telomerase lengthen telomeres

• telomerase enzyme contains a short strand of RNA in its active site

• RNA sequence is complementary to telomere DNA sequence

• telomerase active site recognises and binds to telomere DNA sequence

• the RNA acts as a template to extend the 3’ end of the telomere DNA ⇒ reverse transcription

  • template (RNA) → synthesise (DNA)

• free DNA nucleotides base pairs with RNA template, telomerase catalyse formation of phosphodiester bonds between nucleotides

• telomerase moves to the right and synthesise another repeat

• once the 3’ end is extended, the other strand {5’ end) is then extended in the usual way of DNA replication using primase, DNA polymerase and DNA ligase

introns → structure

• introns are non-coding sequences of DNA interspersed between exons (coding regions)

• splice sites at the ends of each intron serve as signals for RNA splicing

introns → function

1. control gene expression

   • introns may contain regulatory sequences that controls transcription initiation

   • removal of introns regulates export of mRNA from nucleus to cytoplasm

2. regulates alternative RNA splicing → different splice sites are recognised

   • allows one gene to code for more than one type of polypeptide

   • different exons are removed together with introns, resulting in different mature mRNA molecules formed from the same pre-mRNA

   • after translation, this results in different polypeptides arising from the same gene

3. facilitate evolution (formation) of new and potentially useful proteins

   • a single protein is divided into regions called domains, with different structures and functions

   • in many cases, different exons code for different domains of a protein

   • introns increase probability crossing over between alleles of a gene on homologous chromosome during meiosis → leads to useful recombination

   • exon shuffling could lead to new proteins with novel combinations of functions

control elements

• control elements are non-coding sequences

• they help regulate transcription of a gene when transcription factors (protein) bind

promoter → structure

• located 25 nucleotides upstream from transcription start site

• contains TATA box, a short sequence of T and A nucleotides

promoter → functions

• specifies transcription start site on the template strand of DNA

• RNA polymerase and general transcription factors bind to promoter to initiate transcription

  • a general transcription factor (TF) recognise and bind to TATA box in the promoter

  • other general TFs and RNA polymerase are recruited to bind at the promoter, resulting in assembly of the transcription initiation complex (TIC)

enhancers (distal control elements) → structure

• enhancers are DNA sequences located thousands of nucleotides away from a gene

• it can be upstream or downstream of the gene or even within an intron

enhancers (distal control elements) → function

site where specific transcription factors called activators recognise and bind to increase rate of transcription of the gene → regulate transcription of genes

silencers (distal control elements) → structure

• silencers are DNA sequences located thousands of nucleotides away from a gene

• it can be upstream or downstream of the gene or even within an intron

silencers (distal control elements) → function

site where specific transcription factors called repressors recognise and bind to decrease rate of transcription of the gene → regulate transcription of genes