Biochem 7- DNA Chromatin and The Nucleus

what is the primary role of DNA in the cell? how’s it packaged?

stores information to make cellular compoenets- packaged into chromosomes within the nucleus as chromatin

describe the steps of information from DNA-protein (6)

1. RNA polymerase- reads DNA and makes premRNA- has introns and exons

2. introns are spliced out- 5’ methyl G cap and 3’ poly A tail is added- mature RNA

3. mRNA is exported through nuclear pore

4. ribosomes read mRNA in codons of 3 nucleotides- recognised by tRNA that carry the matching amino acids

5. hates at A, P, E in the ribosomal sites

6. protein folds as it exits

what is intergenic DNA and what elements does it contain (3)

intergenic DNA- DNA between the genes- 75%

repetitive elements-

• tandem repeats found in centromeres for mitosis

• interspersed repeats: transposons that cumove for genome evolution- helped drive placenta evolution by carrying the progesterone elements. viruses that inserted themselves into the genome

unique non codon RNA genes- microRNA

how did retrotransposons contribute to placenta evolution?

contained progesterone-responsive regulatory element added near genes involved in early pregnancy- made these genes respond to progesterone which were needed for implantation and placental development

how is DNA packaged into chromosomes?

• 23 pairs- 22 autosomes and 1 sex chromosome pair

• p arm is the small arm and q long arm

• centromere- where the two arms meet

• end of chromomosome- has telomeres- highly repetitive

what are the main functional regions inside the nucleus and what do they do? (6)

1. chromosome territories- each chr. has its own region- RNA pol stays and genes move towards it. genes that are translated together are located in the same place- like NFkB TF are located together

2. nucleolus- ribosomal RNA for protein translation

3. peri nucleolar(Around the nucleolus)- low in mRNA producing genes- have tRNA genes for ribosome production

4. nuclear speckles- splicinng factors

5. nuclear lamina- tissue specific heterochromatin- usually off in most tissues- silence genes

6. nuclear pores- chromatin that are being transcribed- mRNA can come here for translation

what are euchromatin and heterochromatin? what does nuclear lamina do?

1. euchromatin- open and active DNA- genes can be transcribed

2. heterochromatin- condensed, inactive- found at nuclear lamina, silence genes during differentiation

3. nuclear lamino- represses genes and maintains nuclear structures. mutations in this can cause premature ageing as genes aren’t repaired

how is DNA packaged by histones?

DNA wraps- 175bp around histone actamers H2A, H2B, H3, H4 and linker histone H1

• makes nucleosomes which fold into chromatin

• packaging is tight so needs mechanisms for DNA to access it

what is cohesion and how does it function during replication?

cohesin- ring protein complex- holds sister chromatids together

• loaded onto chromosomes in G1 phase before rpelication

• cohesion keeps chromatin aligned as DNA is copied

• after replication cohesion is removed from everywhere but the centromere until mitosis

what are telomeres and how do cells keep them from getting too short? when is it used?

• DNA replicates from 5’ to 3’ and lagging strand needs RNA primers

• primers when removed from the end of chromosomes- the ends shorten and telomere is shortened

• telomerase- RNA dependent DNA polymerase- adds TTAGGg repeats- extends telomeres

• early embryonic development- if teleports are too short, chromosomes are instead. in cancer, proliferation shortens telomeres and results in cell death

how are telomeres protected after being made? (4)

1. shelterin complex- 6 proteins TRF1/TRF2- recognise TTAGGG repeats, POT1 binds the overhang and controls telomerase

2. T loops: end of tolermore loops back on itself to hide the chromosome end and stops DNA machinery from “fixing” the end

3. D loops: single stranded overhand- inserted to the double stranded part of telomere- extra protection

4. G- quadruplex- many guanine repeats- resistant to enzymes that cut DNA

what makes up the histone core and how does it interact with DNA?

• core has H2A/H2B/H3/H4- in tetramers x2

• DNA is negatively charged- histones are positive

• H3/H4- stably associated with DNA

• H2A/B- flexible and can be removed first before H3/H4

what are histone tails and how are they modified? why are histone modifications important? (5)

tails- ends of histones that stick out of the nucleosome

• methylatation- still positive and DNA is bound

• demethylation- still pos and DNA is bound

• acetylation- neutralises the positive charge and allows DNA to move and be transcribed

• phosphorylation on serine

• attachment of proteins like ubiquitin

!!! modify the DNA accessibility to control gene expression and function

what enzymes control histone modifications?(3)

1. writers- add chemical modifications to histone tails

2. readers - bind to specific chemical modifications to influence DNA function

3. erasers- remove modifications to reverse the effect

how was histone movement studied in cells?

• histones fused with GFP

• laser bleaches the fluorescence and then watch the recovery

• if the histones couldn’t move then there’s no recovery but the fluorescence does return- constantly replaced and moving

what happens to histones during DNA damage repair?

double stranded breaks- H2Ax replaces H2A at damage sites

H2Ax is phosprylated and recruits the repair machinery such as RAD51

ensure the DNA repair is proper and stops the cell cycle

what are some histone variants and their functions? (3)

H3.3- found at actively transcribed genes

H2AZ- marks boundaries between euchromatin and heterochromatin

macro H2A- complete silencing of genomic regions

controls which parts of the genomes are active or silent

structure of histones and their features in the nucleosome?

• 3 alpha helices and 2 linker regions

• H3/H4- horseshoe structure and are stable- defines the nucleosome diameter

• H2A/B- more dynamic and can move in and out- need to be removed first

• histones exchange at promotor active regions- allow transcription

• H3.3 and H2A.z- variant histones at promotor sites/DNA repair- may open up DNA for transcription or marking boundaries

how do histones modifications control gene expressions

• controls the chromatin structure- separate DNA into euchromatin open and heterochromatin closed

• histone methylation depends on the site if its on or off- H3K27(lysine 27, H3)- Polycomb repressive to compact chromatin- SILENCING .H3K4 at lysine 4(enhancer)- reader proteins- OPEN. H3K9-OFF(meth at lysine 9) recognised by HP1 and recruits deactylases and turns the gene off)

• histone acetylation- neutralises positive on lysine- DNA is loose- gene activation

• phosphorylation- can recruit proteins

modifications added by writers, removed by erasers and interpreted by readers

how do DNA modification control gene expression?

• add methyl groups -CH3- to cysostine bases- at CpG islands at the promotor regions

• methylation at promotor regions in the DNA- silences the gene as transcription machinery cant bind

• unmethylated promotors- transcription machinery can bind and the gene is active

• recognised by methyl binding proteins- recruit histone deacytlases- and tighten the chromatin(acetylation loosens, deactivation tightens)

how is DNA recognised by proteins? DNA structure and also motifs (3)

alpha helix DNA- major groove with more exposed sites for proteins and minor groove that’s less accessible

— leucine zipper- coiled and has positive helix end- neg charged DNA

— zinc finger- recognises structures

helix turn helix- 2 alpha helices with loops and fits into the major groove