Bioc0007 Where does the glycosidic bond form between a nucleobase and the sugar? C1 of sugar and N1 of pyrimidine or N9 of purine Polynucleotide A polymer consisting of many nucleotide monomers in a chain (DNA or RNA) B DNA No. Bases per turn = 10.5 Helix

Bioc0007 Where does the glycosidic bond form between a nucleobase and the sugar? C1 of sugar and N1 of pyrimidine or N9 of purine Polynucleotide A polymer consisting of many nucleotide monomers in a chain (DNA or RNA) B DNA No. Bases per turn = 10.5 Helix diameter = 20 A Major groove = 13 A Minor groove = 9 A Solid Core Right handed A DNA Dehydrated conditions No. Bases per turn = 11 Helix diameter = 26 A Hollow Core Right handed Z DNA High Saline / Stress / Methylation No. Bases per turn = 12 Helix diameter = 18 A Solid Core Left handed Nucleosome Octamer of histone proteins (H3-H4 tetramer, 2 x H2A-H2B heterodimers) surrounded by 146 bp left-coiled DNA. H1 histone protein "linker histone" binds to DNA in the linker region - helps compact adjacent nucleosomes (to 30nm fiber) Histone Tails Strings of 19-39 amino acids protruding from nucleosome. - H3 and H4 only have N terminus tails Helicases Hexameric ATPases - break hydrogen bonds in double stranded DNA For replication: - Prokaryotes = DnaB - Eukaryotes = MCM Complex Primase RNA polymerase Adds 10 bp primer onto 5' end of single-stranded DNA DNA Polymerase I RNA primer removal and DNA repair (1 subunit) - Removes RNA primer and replaces with dNTPs DNA Polymerase II Enzyme that proofreads the daughter strand of replicated DNA and corrects any base pairing errors DNA Polymerase III DNA replication (holoenzyme = 9 subunits) DNA Polymerase alpha contains primase activity and initiates DNA synthesis by synthesizing an RNA primer, followed by a short string of DNA nucleotides. (4 subunits) DNA Polymerase delta Lagging strand synthesis Also nucleotide and base excision repair DNA Polymerase epsilon Leading strand synthesis Also nucleotide and base excision repair Sliding Clamp ring-shaped protein that holds the DNA polymerase on the DNA strand - Loaded by Clamp Loader using ATP origin recognition complex (ORC) A protein complex that initiates DNA replication at eukaryotic origins DNA error rate 1 in 10,000, but 1 in a billion go unfixed Intrinsic cellular processes that cause DNA damage - DNA replication - rNTPs used instead of dNTPs - Spontaneous hydrolysis of covalent bonds (depurination, depyrimidation, deamination etc) - Metabolic biproducts (e.g. O2- in H2O2 breakdown) External factors for DNA damage - Environmental (UV, ionisation) - Alkylating agents (add CH3 etc groups to backbone) - Intercalating agents (e.g. EtBr) - Base analogues being incorporated - Genotoxic substances (e.g. Phthalates) Nucleotide Excision Repair (NER) - Eukaryotes 1) XPE, XPC, RAD23B recognise distortions 2) XPB, XPD open DNA around lesion (helicase activity) 3) XPG, RPA unwind and stabilise DNA 4) XPF cuts 5' upstream of lesion 5) XPG cuts 3' downstream of lesion - Gives 24-50 bp region long 6) Gap sealed by DNA polymerase and ligase Single Stranded DNA Repair Mechanisms - Mismatch Repair (MMR) - during and post replication - Base Excision Repair - Nucleotide Excision Repair - Direct Reversal (Prokaryotes) Mismatch Repair (MMR) during replication Complementary strand used as a template Mismatch recognised by DNA Polymerase and finger region moves 3' end into exonuclease region of catalytic palm domain. Mismatch cleaved in 3' to 5' direction, then strand returned to polymerase region of domain. Mismatch Repair (MMR) post replication Complementary strand used as a template Prokaryotes = MutS proteins, Eukaryotes = MutL proteins and MutS homologs (MSH). 1) Mismatch recognised by proteins (prokaryotes = dam methylation of parent (opposite) strand, eukaryotes = sliding clamp interaction) 2) Endonuclease creates nick at site 3) Exonuclease digests mismatch and beyond 4) Repaired by DNA Pol and Ligase Base Excision Repair (BER) Requires complementary strand 1) DNA Glycosylase recognises damaged base (scans minor groove) 2) Glycosylase flips base and cleaves N-glycosidic bond to remove base - leaves apurinic/apyrimidinic site (AP site) 3) AP endonuclease cleaves abasic nucleotide 4) Repaired and sealed by DNA Pol beta and Ligase Nucleotide Excision Repair (NER) - Prokaryotes Requires complementary strand Removes helical distortions (caused by UV damage etc) of 24-50 bp 1) 2 x UvrA and 2 x UvrB form a tetramer and scans DNA for helix distortions 2) Recognises distortions - UvrA dimer dissociates 3) UvrB dimer - Helical activity unwinds distortion 4) UvrC cleaves distortion 8bp upstream and 3bp downstream 5) UvrD removes cleaved section (helicase activity) 6) Repair by DNA Pol and Ligase Direct Reversal (Only Prokaryotes) Doesn't require complementary strand Enzymes, e.g. DNA Photolyase (photoreactivation) and DNA Methyltransferase (methyl group removal) repair DNA Causes enzyme suicide Non Homologous End Joining (NHEJ) Complementary strand not required In non-dividing cells High error rate 1) Ku protein recognises double stranded break 2) Ku70/Ku80 form heterodimer - bind broken ends 3) Dimers recruit DNA protein kinases (DNA-PKcs) - form DNA-PK complex 4) Complex forms synapse at each side of break 5) Ends processed by endonucleases - forms blunt ends 6) Strands joined by DNA Ligase Homologous Recombination (HR) - Rules Complementary strand required Dividing cells (S or G2 phase) Highly accurate Homologous Recombination (HR) - Presynapsis 1) ATM Kinase activates exonucleases - Creates 50bp 3' overhanging tails at double stranded breaks 2) ssDNA overhangs coated in RPA 3) BRCA2 and RAD54 exchange RPA with RAD51 - forms RAD51 nucleofilaments Homologous Recombination (HR) - Synapsis 1) A RAD51 nucleofilament searches for homologous duplex in sister chromatid 2) Forms temporary bond with complementary region - D loop Homologous Recombination (HR) - Postsynapsis 1) DNA polymerase elongates 3' end of damaged DNA via complementary sequences in D loop 2) RAD51 dissociates and D loop exited 3) DNA polymerase connects break in the opposite strand 4) Ligase seals qRT-PCR (Quantitative real-time PCR) uses a fluorescent dye to quantify transcript abundance - continuous monitoring - Means downstream analysis and processing of DNA cannot be done - High resolution and sensitivity Dye Terminator Sequencing utilizes fluorescent labeling of the chain terminator ddNTPs, which permits sequencing in a single reaction, rather than four reactions as in the labeled-primer method. Laser and detector "read" the fragments on capillary gel electrophoresis. - Provides an Electropherogram Illumina Sequencing 1) Adaptors attached on fragmented DNA 2) Fragmented DNA attached to flow cell via adapters 3) Fragmented DNA forms bridge due to complementarity between fragment on the other end and the flow cell 4) PCR amplification synthesises complementary strand 5) Denatured and repeated - forms clusters on flow cell 6) Sequencing begins - the 4 labelled dNTPs applied to cell (label stops 3'-OH from working - only one added each time). Photographed, then fluorescent labels removed so next sequence cycle can begin. Pyrosequencing 1) Sequencing beads, coated in millions of copies of a single DNA sequence/fragment, exposed to reaction mixture containing a dNTP (not dATP - replaced by dATP(alpha)S), ATP sulfurylase, adenosine 5'-phosphosulfate, luciferase, luciferin, DNA Polymerase etc. 2) Addition of a nucleotide releases pyrophosphate. This is used by ATP sulfurylase with adenosine 5'-phosphosulfate to create ATP. ATP drives luciferin to oxyluciferin by luciferase, producing light. 3) Light recorded by CCD camera - intensity proportional to amount of light released. 4) Unincorporated dNTP and Apyrase (enzyme which degrades ATP and unincorporated dNTPs) are removed 5) Next dNTP added 6) Steps repeated in cycles. In Vivo CRISPR editing Lipofection Microinjection Electroporation Viral Vectors Nanoparticles Can also do Ex Vivo gene editing (extract cells from organism, do CRISPR on them, put cells back in organism). CRISPR advantages Accurate Cheap Easy to use Well characterised CRISPR uses Make animal models Reprogram transcription factors and epigenetic markers Understand genetic disease Engineer embryos Allow genetic engineering Do the other CRISPR Cards! And Eukaryotic Termination Models! Transcription Bubble Size 14bp Eukaryote RNA Polymerases - RNA pol I: transcribes rRNA genes (except 5S rRNA gene) - RNA pol II: transcribes genes encoding mRNAs and some non-coding mRNAs-- can NOT bind to promoter itself, complex set of 10 proteins - RNA pol III: transcribes tRNA, snRNA, 5S, rRNA genes Bacterial RNA Polymerase subunits - Two alpha subunits = scaffolding - Beta ' = DNA binding region of core - Beta = rNTP binding region of core - Omega = Scaffolding (not known really) Sigma Factors (Prokaryotes) Protein that binds to RNA polymerase, allowing the complex to bind to and stimulate the transcription of genes - Different tissues have different sigma factors = Allows spatiotemporal expression (due to binding to different consensus sequences). Sigma Factor 70 Domain 1 Binds Discriminator (start point to -10) Sigma Factor 70 Domain 2 Binds -10 region (-10 to -16) - Separates DNA double helix and stabilises ssDNA Sigma Factor 70 Domain 3 Binds 2bp -10 region extension (-16 to -18) Sigma Factor 70 Domain 4 Binds -35 region (6bp) - Forms DNA backbone binding motif (helix-turn-helix) - Allows flexibility for optimum positioning 3.2 region prokaryote transcription Loop between Sigma Factor 70 Domains 3 and 4 - Positions oncoming rNTPs UP element a sequence in bacteria adjacent to the promoter, upstream of the -35 element, that enhances transcription - Binds C-terminal domain (CTD) of RNA polymerase Core promoter elements TATA box = Binds TBP (TATA binding protein) on TFIID BRE = TFIIB B recognition element INR = Initiator element. Binds TBP-Associated Factors (TAFs) on TFIID DPE = Downstream promoter elements. Binds TAFs on TFIID Eukaryotic Pre-initiation complex formation 1) TBP of TFIID binds TATA box - associates RNA Pol II with promoter 2) TBP partially unwinds DNA (not helicase) - bends DNA 3) TAFs bind other control elements (DPE, INR) 4) TFIIA joins - stabilises DNA/complex interaction 4) TFIIB joins - binds BRE to active site of RNA Pol II - Forms early transcription complex 5) TFIIF joins - fully recruits RNA Pol II 6) TFIIH joins - XPB subunit (helicase) unwinds startpoint = Pre initiation complex Mediator Complex ~22 enzymes - Promote initiation complex assembly and mediates response to enhancer regions Order of complexes in transcription initiation (eukaryotes) Closed pre-initiation complex - Open pre-initiation complex - Initiation complex - Elongation complex (once cleared promoter). Rate of transcription 20-50 bp per second (in a processive manner) Rho-dependent termination Hexameric ATP-dependent helicase travels along DNA/RNA duplex and makes contact with RNA Polymerase. Either: - Conformational change in RNA Pol - Pushes RNA Pol forward and off RNA - Pulls RNA out of RNA Pol active site What activates Rho in Rho-dependent termination C-rich areas of DNA Intrinsic transcription termination Stem-loop hairpins due to 8-10 Uracils in a row on RNA. Weakly binds to DNA section, forming loop. This pulls RNA from the RNA Polymerase. What does FACT protein do Facilitates Chromatin Translocation. Temporarily remove a H2A-H2B dimer to allow RNA Pol II to pass. lid, zipper, rudder Hold coding strand away from template strand during transcription in RNA Polymerase RNA Polymerase I rRNA (apart from 5s rRNA) RNA Polymerase II mRNA, snRNAs, siRNAs, miRNAs RNA Polymerase III tRNAs, 5srRNA and others When does capping of mRNA occur? As soon as the 5' end of pre-mRNA is free of the RNA polymerase (20-30bps have are synthesised) CTD importance on RNA Polymerase II - 5' Capping - Partially phosphorylated - recruits 5' capping complex - Splicing - Further phosphorylation - recruits splicing machinery - Polyadenylation - CTD dephosphorylation at serine (5) recruits 3' end processing complex to cut around AAUAAA. 5' capping process 1) RNA 5' triphosphatase - removes gamma phosphate from 5' nucleotide 2) Guanyltransferase - adds GMP to 5' end 3) Guanine-7-methyl-transferase - Methylates guanine Intron recognition sites - 'GU' at start - 'AG' at end - Polypyrimidine tract - Branch point nucleotide Spliceosome assembly Composed of small nuclear ribonucleoproteins (snRNPs - 100 to 300 nucleotide RNA) - U1 binds 'GU' - BBP binds Branch point nucleotide (also U2?) - U2AF65 binds polypyrimidine tract - U2AF35 binds 'AG' U4,U5,U6 then replace the U2 and BBP subunits mRNA rearrangements release U1 and U4 Transesterification reactions can then occur to release the intron lariat. Intron and Exon Enhancer Sequences (ISE and ESE) Strengthen interaction between U1 and 'GU', and U2 and 'AG' = Promotes spliceosome splicing ability. Silencer sequences mask splice sites and block spliceosome assembly. Which enzyme adds Poly(A) Tail Poly(A) polymerase adds ~200 adenosine triphosphates. Purpose of Poly-A tail - Prevents degradation by exonucleases (more stable) - Allows export to ribosomes in cytoplasm (from nucleus) - Terminates transcription - Helps initiate translation (via Poly(A) binding proteins) Examples of rRNA/tRNA precursor RNAs E.coli - 3 rRNAs and multiple tRNAs on same precursor S.cerevisiae - 3 rRNAs but no tRNAs on same precursor Advantage of being on same precursor = same amount of each smaller RNA made. RNase H removes RNA primers in eukaryotes (endonuclease) RNase T removes nucleotides from the 3' end of the precursor rRNA or tRNA (exonuclease) RNase P An endonuclease that cleaves the 5' end of pre-tRNAs. RNase III performs excision of bacterial rRNAs (cuts out the rRNAs from precursor molecule). rRNA excision and modification Excision = by RNaseIII Modifications = by Small Nucleolar Ribonucleoproteins (snRNPs) tRNA excision and modification 1) Intron removed (by SEN endonucleases) - Reveals anticodon loop 2) Forms two half tRNAs - joined by RNA ligase 3) RNase P cleaves 5' end 4) 3' Uracils cleaved and replaced by CCA (by CCA enzyme) 5) Multiple bases modified. RNA Extraction Techniques Column Based = High purity Phenol Chloroform = Quick, Cheap, High Yield Bead Based = Very Quick, High Yield Northern Blot Similar technique [to Southern], except that Northern blotting involves radioactive DNA probe binding to RNA (that has been electrophoresed and transferred to a membrane) Nuclease Protection Assay DNA or RNA probes added to mixture of RNA - forms dsRNA (RNA-RNA or RNA-DNA) complex, resistant to degradation by nucleases for single strands (e.g. S1). Polycistronic One promoter controls many genes' transcription (one mRNA encoded multiple proteins) LacA Thiogalactoside transacetylase - rids cell of toxic thiogalactosides which are also transported across the membrane How do activators stimulate transcription? Cause DNA to bend - activation domain binds mediator proteins which cause RNA Polymerase (II) to begin transcription How is eukaryotic transcription regulated? - Pre-initiation complex - Transcription factors binding to proximal control elements - Distal control elements and enhancer regions (activator proteins) - Tissue specific regulatory transcription factors - Processing of mRNA (Capping, Poly(A) can reduce degradation, splicing regulates which protein is produced) What organisms have DNA methylation? Most eukaryotes - Lost in worms, flies, yeast Bacteria - lack of methylation = foreign DNA Where does methylation of DNA occur? CpG islands (Forms 5-methyl cytosine) (regions at promoter of up to 60% CpG doublets) Importance of DNA methylation (eukaryotes) - Repress promoter activity - Regulate alternative splicing - exons = more methylation - Repress transcription of repeat sequences - Repress translocon relocation - Control X-chromosome activation and imprinted gene expression How does methylation at promoter repress transcription? Methyl-CpG-Binding proteins (with MBD) bind CpGs. These recruit HDACs (Histone Deacetylases) and chromatin remodeling factors, condensing the chromatin. DNA Methyltransferase 1 (DNMT1) Responsible for Maintenance Methylation (conservation of methylation after DNA replication) - Adds methyl groups to unmethylated strand DNA Methyltransferases 3a and 3b Responsible for De Novo Methylation (in gametogenesis and embryogenesis) How many imprinted genes are in humans? ~100 (1-2% of genome) Insulin-like growth factor 2 Major prenatal growth factor - imprinting (expressed by paternal gene). Beckwith-Weidmann Syndrome IGF2 expressed from both maternal and paternal alleles (due to incorrect imprinting) = Overgrown baby or prenatal death Silver Russell Syndrome H19 expressed from both maternal and paternal alleles (due to incorrect imprinting - IGF2 not expressed) - Children with inhibited growth (growth retardation, small stature etc) What histone residues are methylated? Lysine or Arginine What histone residues are acetylated? Lysine What histones can be methylated? H3, H4, and H1(b) How does histone acetylation affect chromatin? Loosens chromatin due to weakening histone/DNA interactions - = Increased transcription Histone acetyltransferases (HATs) and histone deacetylases (HDACs) Add and remove acetyl from histones Bromodomain Protein domain that binds acetylated lysine residues (on histones) - Histone Acetyltransferases have these Histone Methyltransferases (HMTs) Histone modifying enzymes that add methyl groups to lysine or arginine residues histone demethylases (HDMTs) Remove methylation from histones SET domain Protein domain with methyltransferase activity (present in histone methyltransferases) Chromodomain Reads histone methylation - Associated with chromatin remodelling. - 60 amino acids residues (conserved) How does methylation effect histone charge? It doesn't EZH2 - Synthesises histone methyltransferases (is a methyltransferase itself) - Perhaps reorganises histone modifications in daughter cells to how they were in parent cell Missense mutation Point mutation resulting in a different amino acid being encoded Nonsense mutation A mutation that changes an amino acid codon to one of the three stop codons, resulting in a shorter and usually nonfunctional protein. Frameshift mutation mutation that shifts the "reading" frame of the genetic message by inserting or deleting a nucleotide How does wobble occur? Large gap between nucleotides 33' and 34' at 5' end of anticodon. This allows space for non-standard matches. Shine-Dalgarno sequence (AGGAGG); initiates prokaryotic translation 8bps up from AUG f-MET start codon. Binds 16s subunit of prokaryotic ribosome. What prevents the 50s ribosomal subunit binding the 30s in the cell? Initiation factor 3 bound to the E site of the 30s subunit Where do Initiation factor 2 (with GTP) and 3 bind? Amino-acyl site of 30s ribosomal subunit. When is initiation factor 3 displaced? Binding of fMET-tRNA and mRNA - allows 50s subunit to bind. GTP then hydrolysed, releasing IF2 and IF1. EF-Tu (-GTP) Mediates aminoacyl-tRNA binding in A site of ribosome (prokaryotes) EF-G (-GTP) Catalyses translocation of tRNA and mRNA down ribosome (prokaryotes) Release factors 1 and 2 Bind to stop codon - Resemble tRNA Release factor 3 Ensures stop codon correctly recognised and dissociates RF1 and 2 Prokaryotic ribosomal subunits 30s: - 16S - binds Shine-Dalgarno 50s: - 5S - joins small and large subunits - 23S - performs peptidyl transferase reaction Eukaryotic ribosomal subunits 40S: - 18S - binds Kozak 60S: - 5S - joins small and large subunits - 28S - performs peptidyl transferase reaction - 5.8S - Unsure Eukaryotic pre-initiation and initiation translation complexes 43S pre-initiation, 48S initiation, 80S initiation Eukaryotic other elongation factors EF1alpha (+GTP) - escorts amino-acyl tRNAs to A site. GTP hydrolysis moves C-terminal end (carrying amino acid) of A site tRNA closer to P site tRNA for peptidyl transferase reaction EF2 (+GTP) - hydrolysis of GTP translocates ribosome a codon along. Eukaryotic release factors eRF1 and eRF3 (+GTP) - form a complex that binds to A site. - GTP hydrolysis cleaves peptidyl tRNA from ribosome. Tetracycline blocks binding of aminoacyl-tRNA to A site of 30s subunit Streptomycin prevents the transition from initiation complex to chain elongation; also causes miscoding Chloramphenicol Blocks peptidyltransferase reaction at 50S ribosomal subunit Erythromycin Binds to the 50S subunit E site and inhibits ribosome translocation (prokaryotes) Nuclear Import Signal Sequences - Lysine and Arginine - Not necessarily at end of polypeptide Mitochondria Import Signal Sequences - Lysine and Arginine rich - At N terminus (usually) - Amphipathic alpha helix ER Import Signal Sequences - Hydrophobic amino acid rich (1 or so positively charged, followed by 6-12 hydrophobic) - At N terminus Karyopherins importins and exportins (nuclear transport receptors) FG repeats Short amino acid repeats on nucleoporins (in nuclear pore complex) for binding of nuclear transport receptors Are proteins folded before entry into nucleus? Yes Mitochondrial localisation signal (MLS) Amphipathic Alpha helix - Alternating positively charged and hydrophobic residues - Near the N terminus Are proteins folded when transported into mitochondria? No TOM Complex - Translocase of the Outer Membrane - Recognises the MLS - Inserts unfolded protein into intermembrane space (and associates with TIM23 complex) TIM Complex - Translocase of the Inner Membrane - Translocates polypeptide into matrix ATP uses for mitochondrial protein import - Electrochemical Membrane Potential - moves polypeptide through TIM complexes - Cytosol hsp70 chaperone removal - Mitochondrial hsp70 function - translocation through TIM complexes - Redox potential - e.g. Mia40 in intermembrane space SAM Complex - Sorting and Assembly Machinery Inserts outer membrane-bound mitochondrial proteins from intermembrane space. Inner mitochondrial membrane protein insertion - 4 methods Avoiding matrix insertion: - Stop transfer sequence (hydrophobic - behind N terminal MLS) stops TIM23 translocating into matrix - inserted. - TIM22 recognises internal MLS and inserts transmembrane protein Matrix insertion: - 1st signal sequence (MLS) - inserts into matrix via TIM23 and cleaved. 2nd signal sequence used by OXA complex to insert into inner membrane Matrix synthesis: - Inner membrane localisation signal used by OXA to insert into inner membrane. Bioanalyzer A machine which quantifies the size range and quality of DNA or RNA in a sample. TapeStation Machine that gives info on nucleic acid, size, quantity, integrity (not sequence). What primers are added during reverse transcription? 2 sets: - Oligo-T nucleotides - binds Poly(A) tail of RNA - 6bp random hexamers - for cDNA replication RT-PCR 1. All mRNA from sample isolated and converted to cDNA with reverse transcriptase 2. PCR using primers specific to target sequence 3. Visualise with electrophoresis 5' RACE Addition of the cDNA primer straight away to the KNOWN region of mRNA Reverse transcriptase and dNTPs allow reverse transcription in 3' direction Terminal transferase and dATPs allow Poly(A) tail to be added on end of the this 3' growing strand The RNA:cDNA can then be denatured and RNA degraded Oligo-T primers added to the Poly(A) tail, and dNTPs and DNA polymerase extend the cDNA. PCR amplification. 3' RACE Oligo-T primers are added to anneal the Poly(A) tail of the mRNA. Reverse transcriptase and dNTPs extends the cDNA in the 3' direction. The cDNA:RNA complex is denatured and the RNA degraded A primer corresponding to the KNOWN region of the cDNA is added. dNTPs and DNA polymerase extend this strand from the primer in the 3' direction. PCR amplification can be performed on this double-stranded cDNA. RNA-seq 1) Fragmentation and end repair of total RNA 2) Polyadenylation 3) cDNA synthesis and adapter addition (and barcodes) 4) Purification 5) PCR amplification 6) Next generation sequencing used Exome sequencing Sequences only exons: 1) DNA fragmented, linkers attached to each end of fragments 2) PCR amplification 3) Biotinylated primers to all known exons added 4) Exposed to streptavidin beads - binds biotin tightly 5) Extraction and sequencing of exons Deep sequencing Sequencing of a genomic region multiple times. Allows researchers to detect rare RNA or DNA species that comprise of less than 1% of the sample. Argonaute/Piwi proteins Ribonucleases that process interfering non-coding RNAs (siRNA, miRNA, piRNA) and carry them to their target. Dicer argonaute protein Cleaves and loads siRNA Drosha argonaute protein Cleaves and loads miRNA PIWI protein Cleaves and loads piRNA How do interfering RNAs reduce gene expression? (3 ways) - Cleave target RNA - Repress translation and degrade target RNA - Form heterochromatin at DNA where target RNA is transcribed RISC complex RNA-induced silencing complex = Argonaute/PIWI protein + small non-coding RNA Short hairpin RNA (shRNA) Artificial RNA interference (RNAi). Targets specific RNA products to silence specific genes. Created by shRNA libraries, using these to transfect or transduce other cells and insert the gene for the shRNA production What is SRP (signal recognition particle) made of? RNA (1) and proteins (6) What powers the assembly of the translocon complex and the release of the SRP/SRP receptor? GTP Signal Peptidase An enzyme that removes the signal sequence of a polypeptide chain by proteolysis. Type 1 membrane protein N terminus in ER lumen Type 2 membrane protein C terminus in ER lumen What residues does N-linked glycosylation occur on at the ER? Nitrogen of asparagine residues (N-X-S/T) N-linked glycosylation enzyme Oligosaccharyltransferase N-linked glycosylation importance (there are 4) - Resistant to protease degradation - Signals protein is folded - Pathogen protection - Signaling/transport BiP protein A hsp70 ATPase (chaperone) in Sec62/63 pathway - helps pull protein through Sec62/63 complex co or post-translationally. Lectin Carbohydrate binding protein - Binds carbohydrates on proteins in ER lumen - recognises them as aberrant E3 Ubiquitin ligase Polyubiquitinates aberrant protein - labelled for proteosome N-Glycanase Enzyme which removes oligosaccharides chains from ER proteins that have been retro-translocated into the cytosol. (By cleaving amide bond between N-acetylglucosamine and asparagine). ARF protein The coat-recruitment GTPase responsible for both COPI coat assembly and clathrin coat assembly at Golgi membranes. (uncoating of clathrin coat uses Hsp70 ATPases). Sar1 protein Coat-recruitment GTPase responsible for COPII coat assembly on ER membranes Dynamin GTPase Assembles as a ring around the neck of a vesicle, hydrolyses GTP to help with pinching off of the vesicle Rab GTPase Proteins that provide specificity of vesicle trafficking during targeting to acceptor membrane - Bind to tethering protein on target membrane surface NSF (N-ethylmaleimide sensitive fusion protein) An AAA ATPase - Recycles Cis-SNARE complex What specifies membrane bindings? - Rab protein type - SNARE protein types - Phosphoinositide types (determined by PIP phosphatases and kinases in the tissue) KDEL and KKXX signals Present on resident ER proteins at C-terminal end - Allows for retrograde transport back to ER Endoglycosidase H (endo-H) Experimental tool for seeing how developed carbohydrate chain is. O-linked glycosylation involves addition of the oligosaccharide to the oxygen atom on the hydroxyl group of certain serine or threonine residues Done in Golgi Glycosyl transferase Used for O-linked glycosylation Two models of Golgi transport 1. Cisternal Maturation Model 2. Vesicles Transport Model Mannose-6-phosphate Lysosomal targeting sequence attached to peptides in the Golgi - Taken via clathrin-coated vesicles to endosome from trans-Golgi Mucolipidosis II (I-cell disease) Defect in an enzyme to add mannose-6-phosphate to peptides in Golgi FDNPVY LDL receptor signal YXXO EGF endocytosis signal ESCRT Proteins Endosomal machinery to result in intralumenal vesicularisation of ubiquitinated receptors and their ligands EGF internalisation Via EGF receptors. Taken via vesicles to early endosome. Enters into intralumenal vesicles in late endosome, which fuses with lysosome. EGF and receptor are degraded. Importance of NADPH with ROS Reducing (proton donor) - eventual reduction and neutralisation of the ROS Protooncogene A normal gene which, when altered by mutation, becomes an oncogene that can contribute to cancer (via gain of function mutation) Tumor Suppressor gene A gene whose protein products inhibit cell division, thereby preventing uncontrolled cell growth (loss of function mutation can cause cancer) c-Src Protooncogene - encodes a tyrosine kinase Human homolog to v-Src in chickens stem cell cancers - Can initiate new tumors - Highly proliferative (due to mutation) - Can divide indefinitely - Drug resistant - can cause tumor regrowth Main effects of oncogenes - Cell Growth - Cell Proliferation - Cell differentiation - Cell Apoptosis Oncogenic transformation mechanisms - Gene amplification = more protein produced - New promoter = more protein produced - Mutation in control element = more protein produced - Mutation in oncogene - may cause hyperactive or degradation-resistant protein v-sis oncogene Oncogene of Simian sarcoma virus. Produces PDGF-like protein in cells, causing repeated activity of tyrosine kinases, causing cell proliferation. v-ErbB An oncoprotein in avian erythroblastosis virus (AEV). Produces just kinase domain of EGF receptor (erbB1 tyrosine kinase), so constitutively active tyrosine kinase domain. Causes cell proliferation. c-erbB Human oncogene (neu = rat oncogene). Point mutation in eerB1 receptor (EGF receptor) at transmembrane domain. Dimerises receptor so constitutively active. Causes cell proliferation. EGF receptor Oncogenic capabilities -Proto-onco: ERBB1 (EGFR) and ERBB2 (HER) -Mode of activ. in tumor: Mutation and amplification -Assoc. tumor: Lung adenocarcinoma and breast carcinoma Induce proliferation via constitutively active tyrosine kinase domains. Onc-Met Oncogene of the protooncogene c-Met (Hepatocyte growth factor receptor (HGF receptor)). Looses transmembrane domain, so becomes cytosolic. Aberrantly activated. Rho GTPases A subfamily of Ras monomeric G proteins, which includes Rho, Rac and Cdc42, that stimulate formation of various actin-containing structures within cells. Turn off and on signal transformation pathways (via associated-GTP hydrolysis). Rho-GTPase mutations can cause... - Loss of cell junctions - Loss of polarity - Increased cell mobility These can cause intravasation, and multilayering of cells. matrix metalloproteinases (MMPs) Digest extracellular matrix, including cell-matrix and cell-cell adhesions (CD44 and E-cadherin destruction). - Also growth factor release MMP activation In trans-Golgi. Furin proteins cleave propeptide domain of MMP, activating it. Apoptosis activation factors DNA damage, stress, starvation, oncogene activation, tumor suppressor inactivation. Apoptosis inhibitors Bcl-2, PI3K, Akt Bcl-2 Oncogene that regulates apoptosis Rb Tumor suppressor. Phosphorylated by S cyclin/ cdk2 complex, causing inactivation, allowing cell cycle to progress. - If Rb is mutated, it becomes inactivated, meaning cannot form inhibitory complexes and cell cycle not regulated. Warburg effect -use of glycolysis under normal oxygen conditions (aerobic glycolysis) -allows products of glycolysis to be used for rapid cell growth -activated by oncogenes and mutant tumor suppressors Tumor M2-PK Upregulates biosynthetic processes (nucleic acid, amino acid, phospholipid synthesis), and prevents entry of pyruvate into mitochondria, therefore reducing oxidative phosphorylation. Unconventional nutrients used by cancer for metabolism: - Acetate - increased acetyl CoA production - Ketone bodies - Converted to acetyl CoA for metabolism - Lactate - Converted by LDHA and LDHB to pyruvate - Ammonia - Enters Krebs cycle for use - Extracellular proteins - Entry and recycle via pinocytosis Where does mTOR get information from - Growth hormones (EGF, IGF, VEGF) - Nutrients (increase = more activity) - ATP:AMP ratio (more ATP = more activity) Effect of mTORC1 Protein, nucleotide, lipid synthesis Cell growth Metabolism Effects of mTORC2 Cytoskeletal rearrangements Proliferation via IGF receptors Rapamycin mTOR inhibitor Myc upregulation by: - Enhancer binding to and activating promoter region - Gene amplification - Aberrant upstream signaling (e.g. overactivity of mTOR) Myc upregulation effects: - Upregulates all aerobic glycolysis enzymes = increased glucose metabolism - Upregulates glutaminolysis - Upregulates folate cycle = nucleic acid synthesis - Upregulates lipid, protein, reducing power synthesis What protein does Myc bind to to become a transcription factor? Max What degrades Myc? Polyubiquitination of lysine 48 sends it to proteasome. HIF1 Example of proteosome mediated hydrolysis; checks O2 levels; under normal conditions is degraded via hydroxylation then ubiquitination; under oxygen stress, not hydroxylated; associated with VEGF and neovascularisation. IDH1/2 Converts isocitrate to alpha-ketoglutarate. Mutation produces oncometabolite R-2-hydroxyglutarate instead. - IDH1 = primary NADPH producer in most tissues. R-2-Hydroxyglutarate effects - Inhibits HIF prolyl-hydroxylase = overexpression of HIF1 = lots of VEGF released - Inhibits histone and DNA methylases Reactive Oxygen Species (ROS) cellular importance Cell division and differentiation Apoptosis regulation Ion homeostasis Cell signaling pathways NADPH Oxidases (NOX) Catalyse NADPH-dependent reduction of oxygen to superoxide, which can react to form hydrogen peroxide. - Stimulated by starvation, growth factors, and hypoxic stress. Pentose Phosphate Pathway (PPP) produces NADPH and ribose 5-phosphate for nucleotide synthesis - Reduces ROS Super Oxide Dismutase Take free radical O2- and add hydrogens to make H2O2 hydrogen peroxide O2---H2O2 Antioxidant defences - Enzymes - Catalase, Super oxide dismutase, GPx (peroxisomes) - Glutathione - Vitamins A, C, E NRF2 Regulates genes for enzymes of glutathione meatbolism, NADPH production, iron metabolism, etc (regulates antioxidant response) - Upregulated in cancer cells = cancer cells can tolerate oxidative environments Nrf2 Inactivation In cytosol, binds to Keap1. This binds to Cullin 3 ubiquitin ligase. Ubiquitinates Nrf2, so taken to proteasome for degradation Nrf2 activation Oxidative environment = Cysteines on keap1 get oxidised, so dissociates Nrf2. Nrf2 enters nucleus and associates with Maf protein, acts as a transcription factor. How is CoAlation of proteins protective? CoAlation of cysteine residues on protein prevents oxidation by ROS of the protein. CoA production Made of ATP, Pantothenate, and cysteine - Requires Coenzyme A Synthase - Found to have mutations in neurodegeneration CPSF Cleavage and polyadenylation specificity factor. Binds AAUAAA on pre-mRNA CsTF Cleavage stimulation factor. Binds GU-rich element of pre-mRNA to initiate cleavage. Rat1 Exoribonuclease. Degrades uncapped RNA in 5' to 3' direction. Involved in transcription termination in eukaryotes. When it runs into RNA Pol II, it causes the polymerase to fall of DNA and end transcription. Importance of Pre-mRNA processing Ribonucleoproteins Composed of RNA and proteins -involved in intron excision and splicing and other aspects of RNA processing