DNA to protein

DNA supercoiling, histones and chromatin

DNA wraps around histones - coils around positively charged histone proteins, forming nucleosomes

histones are rich in lysine and arginine which help them bind tightly to the negatively charged DNA

nucleosomes are further coiled and folded into a thicker structure - 30 nm chromatin fibre which forms a spiral structure

supercoiling forms heterochromatin - using histones

DNA length is reduced by 40,000 times → chromatids which come together to form a chromosome

DNA structure

• pentose sugar (deoxyribose)

• nitrogenous base - attached to the '1’ carbon on the pentose sugar

  • purines (A, G)

  • pyrimidine (T, C)

• phosphate groups - attached to the 5’ carbon atom on the pentose sugar

DNA replication

DNA helicase unwinds the double helix by breaking the hydrogen bonds between base pairs, single-stranded proteins bind to the separated strands to prevent reannealing, topoisomerase II (DNA gyrase) prevents supercoiling and relieves tension

DNA polymerase alpha (primase) synthesises a short RNA primer to provide a 3’ OH group for DNA polymerase to begin adding nucleotides

DNA polymerase epsilon adds nucleotides 5’ → 3’ direction on the leading strand

the lagging strand runs the other way - DNA polymerase delta adds short DNA sections (okazaki fragments)

RNase H (exonuclease) removes the short RNA primers and DNA polymerase delta replaces primers to fill the gaps

DNA ligase seals the gaps by forming phosphodiester bonds to complete the sugar-phosphate backbone

types of DNA polymerases

alpha: starts replication, works with primase to synthesis a short primer - low fidelity

delta: synthesises the lagging strand - high fidelity

epsilon: synthesises the leading strand and has strong proofreading ability - high fidelity

DNA to RNA

RNA chains are make by connecting RNA nucleotides together in the same way as DNA except as a single strand

pyrimidine is uracil (instead of thymine)

transcription requires DNA polymerase

transcription requires a pre-initiation complex which positions the RNA polymerase in a place ready for transcription

initiation of transcription and its regulation

promoter and core promoter sequence

RNA polymerase

general transcription factors

chromatin remodelling

activation and repressors

promoter and core promoter sequence

promoters: region of DNA that promotes transcription, lie upstream from the transcription start site

core promoter sequence: lies within the promoter e.g. TATA BOX

RNA polymerase binds a core promoter in the presence of transcription factors

RNA polymerase

RNA polymerase I: synthesises rRNA

RNA polymerase II: synthesises mRNA

RNA polymerase III: synthesises tRNA

general transcription factors

TFIID (with TBP) binds to TATA box in the promoter

TFIIA and B join, stabilising the complex

RNA pol II arrives via TFIIF

TFIIE and H are recruited

TFIIH unwinds DNA and phosphorylates RNA pol II → initiates transcription

chromatin remodelling

dynamic structural changes to chromatin that make DNA more/less accessible for transcription, and are crucial for gene activation or repression

histone modification:

• acetylation (histone acetyl transferases - HATs): add acetyl groups to lysine residues on histone tails - neutralises positive charge, loosens DNA-histone interaction → opens chromatin

• deacetylation (histone deacetylases - HDACs): remove acetyl groups → tightens chromatin, represses transcription

• methylation (histone methyltransferases - HMTs): methyl group added to lysine or arginine residues - doesn’t change the charge of histones - alters the chromatin structure and accessibility of DNA to cellular machinery → represses transcription

DNA methylation

DNA methylation: addition of methyl group to cytosine bases in CpG dinucleotides - regulation of gene expression without changing DNA sequence

hypomethylation: loss/reduction of methylation → genomic instability (vulnerable to mutational damage), oncogene activation (lead to cell proliferation and cancer)

hypermethylation: excessive methylation at previously unmethylated sites such as promoter regions → transcriptional silencing (such as tumour suppressor), disruption of normal cellular regulation

activators

bind DNA sites called enhancers which can be located far away from a promoter - they increase the affinity of RNA polymerase to its promoter, speeding up transcription. looping of DNA brings enhancer in close proximity with the promoter, facilitated by transcription factors and co-regulators - recruits other transcription factors e.g. co-activators with HAT activity

co-repressors

can block transcription

a repressor transcription factor binds to a specific DNA sequence (at silencers) → recruits co-repressors to the site → recruit HDACs or block the assembly of the transcription initiation complex

co-transcriptional mRNA processing

prior to protein synthesis

conversion of pre-mRNA into mature mRNA:

• capping

• polyadenylation

• splicing

capping

5’ processing

capping of the pre-mRNA involves the addition of 7-methylguanosine at its 5’ OH end to the 5’OH end of the nucleotide forming an unusual 5’-5’tri-phosphate linkage (5’cap)

protects the 5’ end of the primary RNA transcript form ribonuclease attack

helps position the mRNA on the ribosome ready for translation

polyadenylation

3’ processing

AAUAAA is recognised and cleaved by a ribonuclease → a poly(A) tail added to the 3’ end of a newly synthesised mRNA strand using the enzyme polyadenylate polymerase

regulates its transport to the cytoplasm via the nuclear pore

provides stability and protection to the mRNA from enzymatic degradation in the cytoplasm

RNA splicing

pre-mRNA is made up of both introns and exons

introns are spliced out, leaving the exons for translation

translation

initiation

elongation

termination

structure of a ribosome

40S (smaller unit) which attaches to and reads the mRNA

60S (large subunit) contains A, P and E site which joins amino acids to form a polypeptide chain

tRNA

single stranded RNA which forms secondary hairpin loop-like structures with itself, stabilised by intramolecular hydrogen bonds between complementary bases

contains an anticodon (3 nucleotides) which corresponds to 3 bases of the codon unit on the mRNA

each tRNA is attached to a specific amino acid

initiation

40S subunit binds to initiation factors and a cap-binding complex that recognises the 5’cap of the mRNA

complex scans along the mRNA to find the start codon (AUG)

initiator tRNA (Met-tRNAi) binds to AUG via its anticodon

60S joins to form the full 80S ribosome

elongation

another tRNA with the correct anticodon binds to the next mRNA codon in the A site

peptidyl transferase forms a bond between the amino acids in the P site and A site

the ribosome shifts one codon along the mRNA, the empty tRNA exits from the E site, the growing polypeptide chain shifts to the P site

termination

a stop codon enters the A site → release factors bind (instead of a tRNA) → peptidyl transferase adds a water molecule (instead of an amino acid) → releases the polypeptide, ribosomal subunits dissociate and mRNA is released