Prokaryotic and eukaryotic DNA replication, and

Prokaryote Eukaryote Difference Single origin of replication Multiple origins of replication between • Increase efficiency of DNA replication prokaryotic and Replication occurs in 2 opposing direction • Minimise effects of mutations (ie nonsense) eukaryotic simultaneously • DNA packed in chromatids replication Occurs in the cytoplasm Replication occurs in 1 direction

Occurs in the nucleus High fidelity of • Mismatch base repair (MutL and MutS, DNA polymerase has high affinity for correct bases à 1 in 10^5 errors replication recognises non-methylated newly synthesised • When DNA polymerase encounters an incorrect base pair, it will not undergo (replication) strands) conformational change, therefore, it cannot initiate nucleotide polymerisation

3’ -> 5’ Exonuclease activity à1 in 10^2 errors • Proof-reading system in built in DNAP; if wrong bases are added, DNAP clips off nucleotide; cleavage of phosphodiester bonds supplies energy for polymerisation, allowing DNA replication to occur

Mismatch base repair à 1 in 10^3 errors • MutL and MutS, recognises transient nicks in newly synthesised strands; MutS binds towards mismatch base, MutL scans strand for nick and triggers degradation of newly synthesised strand from nick up to mismatched base Ground state On Off • Activator protein: helps RNA polymerase bind • Tightly compacted chromatid in the nucleus, regulatory sequences required for towards transcription (promoter, promoter proximal regions and upstream activation site/UAS/ • protein: hinders binding of RNA enhancers) are inaccessible à 1) tail acetylation and methylation, 2) SWI-SNF polymerase towards promoter (repressed chromatin remodelling complex, 3) DNA methylation state) o GTF (General Transcription factors) binds towards promoter proximal regions (same for all genes; highly conserved) o Upstream regulatory sequence (enhancers) is specific for each gene à binding site for unique transcription factor • RNAPII requires many regulatory proteins to initiate transcription (binding of TBP to TATA box, GTFs and activator to form pre-initiation complex)

Regulation of Repressor transcription in • Binds to operator and turns gene off • Can be inactivated by an () • Sometimes need a co-repressor to be activated (repressible operon) Activator • Binds to promoter and turns genes on • Can be inactivated by an repressor (repressible operon) Co-repressor • Sometimes required to activate repressor (repressible operon) • ie trp in trp operon à binds towards repressor and switches it on to its active state Inducer • Specific for inducible • Either 1) activates activator 2) inactivate repressor • ie allolactose in à binds towards lacI (repressor) and prevent it from binding to operator • ie cAMP in lac operon à binds towards CAP à complex bind towards CAP binding site Positive regulation • Involves activator • Binding of activator allows gene to be activated • ie Lac operon in the absence of glucose à high cAMP levels à binding of CAP-cAMP (activator) binds to enhance transcription Negative regulation • Involves repressor • Binding of repressor prevents binding of RNAP • Ie Lac operon in the absence of lactose (binding of LacI à repressor) Inducible operon • Example: Lac operon, arabinose operon • Usually catabolic • Normally off (repressor constantly binds + activator does not bind) • Can be turned on when induced (inducer binds to activator à increases binding affinity on DNA binding site/inducer binds to repressor à detached from operator) Repressible operon • Example: trp operon • Usually anabolic • Normally on (activator constantly binds + repressor does not bind) • Can be turned off when repressed (co-repressor binds to repressor to activate repressor à repressor binds to operator) Feedback inhibition • Gene product inhibits activity of enzyme involved in transcribing the gene

Feedback repression (ie trp of trp operon) • Gene product interacts with regulatory protein (ie trp as a ) to repress the gene Transcriptional attenuation (Trp operon) • Availability of end product () determines how fast the leader sequence will be translated à determines whether a hair pin loop structure (transcription of trp gene not completed) or non-terminating loop will form (transcription of trp gene completes) Regulation of Lac operon – inducible operon (produces enzyme that digest lactose) Gene activation by the binding of activator to promoter transcription • Polycistronic operon (codes for 3 proteins, Lac Z, Lac Y, Lac A) (GAL gene, found in yeast à convert galactose to glucose) • Negative regulation • Involves regulation of transcription (mediated by o Lactose absent: LacI (repressor) remains bound to promoter à gene binding of activator on ) inhibited • Regulate gene expression in response to o Lactose present: allolactose (inducer) bind towards LacI à LacI detaches physiological conditions (presence and absence of from operator à gene activated galactose) • Positive regulation • NOTE: transcription of GAL gene is repressed in the o Glucose present: low cAMP (inducer) level à CAP protein (activator) does presence of galactose and glucose regardless of the not bind to promoter à gene inhibited, catabolite repression presence of Gal 4 on UAS à repressor bind o Glucose absent: high cAMP (inducer) level à cAMP forms complex with CAP between UAS and promoter à recruits factor that à bind towards CAP-cAMP binding site à bend DNA to allow better access has HDAC () activity à DNA of RNAP à gene activated becomes deacetylated in this region à transcription turned off à gene inhibited Trp operon – repressible operon (produces tryptophan amino acid) (link) • 1 operon, multiple genes, multiple proteins Gene repression by microRNA • Feedback repression • Noncoding RNAs (RNA interference) produced o Product of a pathway interacts with a regulatory protein (e.g. a repressor) to via transcription à capped and polyadenylated stop transcription of genes encoding enzymes in the pathway to form pre-microRNA à transported to o Tryptophan present: Trp (co-repressor) binds towards repressor à cytoplasm repressor activated à binds towards operator à gene inhibited • In cytoplasm -> cleaved by dicer to form duplex o Tryptophan absent: repressor does not bind à gene activated à 1 strand digested à combines with argonaute protein to form RISC – RNA induced Silencing Complex) • RISC binds towards complementary sequence in genome o Fully complementary: mRNA degrades o Partially complementary: translational repression