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Study Guide for Mohnen portion of EXAM #3 BCMB/BIOL/CHEM 3100 (MWF, Tu breakout; 10;10-11:00 class) Drs. Debra Mohnen and Daniel DerVartanian General list of material you are responsible for for Dr. Mohnen’s part of Exam #3. This includes material on the last part of lipids; nucleic acids; DNA replication; transcription and translation. [NOTE: Exam #3 will cover the last part of the material covered by Dr. Mohnen and the material covered by Dr. DerVartanian. This document is meant only as a guide for your preparation for that part of the material from Dr. Mohnen.] You are responsible for all material covered in the lecture and in the breakout sections, and in the class text, as described by Dr. Mohnen, regarding lipids (relevant sections of Chapters 11, 12, 13) nucleic acids (Chapter 33) , DNA replication (Chapters 34-35), transcription (Chapters 3638) and translation (Chapters (39-40). The topics covered include, but are not limited to the following. The part of the material on lipids that we did not cover on Exam 2. This material starts with the class notes on Lipid rafts and goes through the end of those notes (including the material on these topics in the book). [This includes the following.] Make-up and principals of biological membranes Lipid rafts Three types of membrane proteins (in detail): peripheral, integral and lipid-anchored (3 types) Membrane transport: including passive, primary active and secondary active (and examples of each, in detail) Signal transduction: including G proteins, and detailed examples and understanding of different signal transduction cascades including: common secondary messengers, seven transmembrane helix receptors, adenylyl cycles pathway, inositol phospholipid signaling, receptor tyrosine kinases Structure of DNA and RNA Know and be able to draw structures of 5 bases and their nucleosides and nucleotides for RNA and DNA Where H-bonding occurs between bases Names and abbreviations of bases, nucleosides and nucleotides Understand pyrimidines and purines and the preferred conformation in biological organisms Nucleotides usually complexes in vivo with divalent cation (e.g. Mg++) Nucleotides as building blocks of nucleic acids (process, i.e. nucleophilic attack of 3’-OH on αphosphate of incoming NTP History of DNA: Friedrich Miescher, Fred Griffith, Watson and Crick, Avery, MacLeod & McCarty, Roger Herriott & Alfred Hershey, Chargaff rule DNA double helix: rise = 3.3 Å, pitch = 34 Å, 10.4 bp/turn, 1000 bp = kb Major groove vs minor groove Noncovalent forces that hold helix together A vs B vs Z DNA Different types and sizes of chromosomes Melting point: Tm, melting curve, OD 260 of nucleic acids, ss vs ds DNA DNA is supercoiled in vivo Topoisomerases RNA: rRNA, tRNA, mRNA, small RNA RNAs involved in protein synthesis, post-transcriptional modification & DNA replication, regulatory RNAs Bacterial rRNA:5S+23s+16s; eukaryotic rRNA: 55s+5.8s+28s+18s Hairpins Chromosome packing: nucleosome (2x H2A, H2B, H3, H4) = histone octamers + 146 bp DNA + H1 + 54 bp DNA Packing of DNA: nucleosome (10x); solenoid (4x); protein scaffold (200x) Nucleases: specify 5’ vs 3’ ends, endo vs exo, understand how to denote and read specificity of cleavage RNA is less stable than DNA, RNA sensitive to alkali hydrolysis Restriction enzymes; be able to read and use restriction enzyme tables, blunt end vs stick end Palindromes; use of restriction enzymes DNA Replication: DNA Repair and Recombination Semiconservative vs conservative DNA replication, Meselson & Stahl Arthor Kornberg: discovery of DNA Pol I Function of each of activities of DNA Pol I: 3 activities: 5’→3’ polymerase, 5’→3’ exonuclease; 3’→5’ exonuclease (and functions in vivo!) Synthesis, editing/primer removal, proofreading Klenow fragment processivity Delucia & Cairns experiment DNA PolI vs DNA Pol III, and other DNA polymerases DNA synthesis in E. coli Initiation: OriC Replisome: primosome (primase+helicase), DNA Pol III, ss DNA binding proteins Okazaki fragment, leading vs lagging strand DNA replication: Ori C, Terminator region (tre), nick translation, RNA primer, DNA ligase Difference between prokaryotic and eukaryotic DNA synthesis Telomers, telomerase Sanger Sequencing: (dideoxy sequencing) DNA repair: error rate of DNA synthesis by different enzymes and with repair enzymes direct repair (photolyase) vs excision repair pathway (know pathway) endonuclease/DNA glycosylase removes damages/deaminated bases and gap filled by DNA Pol I Types of DNA damage PCR RNA Synthesis & Regulation in Bacteria; Gene Expression in Eukaryotes; RNA Processing in Eukaryotes Gene: DNA that is transcribed RNAs: rRNA: 80% of RNA, part of ribosomes tRNA: 15% or RNA, 73-95 nucleotides long mRNA: discovered by Jacob & Monod, 1-3% of RNA, unstable small RNA: may have catalytic activity (e.g. U6 & U2 of spliceosome); sn RNA (part of splicing machinery), snoRNA( rRNA biogenesis and modification), microRNA (regulates use of RNA) Small interfering RNA (siRNA): antiviral defense mRNA degradation long non-coding RNA (lncRNA): gene regulation Piwi interacting RNA (piRNA): gene regulation RNAs involved in protein synthesis, post-transcriptional modification & DNA replication, regulatory RNAs E. coli RNA Pol holoenzyme (α2ββ’ωσ) vs core enzyme (α2ββ’ω) E. coli RNA Pol does all three: initiation, elongation, termination Searches for and finds initiation sites (~2000 in E coli), unwinds DNA, finds transcription start site, totally processive, detects termination signals, interacts with activators & repressors σ recognizes promoter; σ70 recognizes promoters of housekeeping genes Consensus sequences in E. coli: TATA box (TATAAT); -35 region (TTGACA) Strong vs weak promoters Coding strand vs template strand and relationship to mRNA Initiation of transcription: holoRNA Pol Elongation: δ leaves and NusA binds Termination: RNA hairpin causes strong pause, some times Rho protein (6mer with ATPase activity) involved Eukaryote RNA Pols: RNA Pol I: rRNA, 18s, 5.8s, 28s RNA Pol II: mRNA, snRNAs (U1, U2, U4, U5) RNA Pol III: t RNA, 5s rRNA, U6 snRNA, small RNA Effect of α-amanitin on different RNA Pols Regulation of housekeeping vs differentially expressed genes Transcription factors: activators vs repressors (both can be allosterically regulated) Repressor: inducer vs corepressor Lac Operon: lacZ (β-galactosidase), lacY (Lactose permease), LacA (thiogalactoside trasnacetylase) Operator region in Lac Operon Lactose vs allolactose, escape synthesis vs induced synthesis Catabolic repression: concept that in presence of Glc PEP-dep sugar phosphotransferase system causes Glc to be phosphorylated to Glc-P However, in absence of Glc, P is transferred to Adenylate cyclase yielding cAMP which activates cAMP regulator protein (CRP) (also called catabolite activator protein, CAP) yields ~50X stimulation of transcription at the Lac operon Eukaryote promoter elements: RNA Pol II promoters: enhancer + TATA box (-24 to-32) + Inr (-3 to +5) enhancer + Inr (-3 to +5) + DPE (+28 to +32) Inr: initiator element; DPE: downstream core promoter element Cis-acting element: DNA sequences that regulate expression of gene located on same DNA molecule Transcription initiation in eukaryotes: TFII: transcription factor for RNA Pol II (TF-D (with TBP),A,B,F (then initiate),E,H TFIIH: opens double helix & phosphorylated CTD of RNA PolII change from initiation to elongation Enhancer: cis-acting element that can be on either DNA strand and stimulate transcription even 1000s of bp away Most eukaryote TFs interact with multiple proteins to give large complexes Mediator: complex of 25-30 subunits interact with transcription machinery before initiation begins; bridge between enhancers and promoter-bound Pol II Steroid Hormones: estradiol as example Estrogen receptor is nuclear hormone receptor; has DNA binding domain (with zinc-finger domain) and ligand binding domain Binding of estrogen receptor with estrogen allows coactivator to bind nuclear hormone receptor that is bound to DNA of specific genes Coactivator: protein that binds to receptor only after is has bound ligand; coactivators can stimulate transcription by loosening interaction between histones and DNA, making DNA more accessible to transcriptional machinery Histone acetyltransferases (HATS) acetylate histones; this reduces affinity of histones for DNA and generates docking site for transcription factors that have Bromodomains (domains that bind to acetylated histones and acetyllysine) Bromodomains are present in chromatin-remodeling machines (ATP-powered complexes that make DNA in chromatin more accessible) Histone deacetylases can remove acetyl groups resulting in inhibition of transcription Processing of RNA: cleavage, modification of nucleotides, addition of nucleotides Both prokaryote and eukaryote rRNA transcripts contain multiple rRNA and tRNA genes that must be removed from the primary transcript by endonuclease action Eukaryote pre-rRNA is first undergoes nucleotide modification prior to cleavage of the individual 18s, 5.8s and 28S rRNA For RNA Pol III: RNaseP (ribonucleoprotein, 377 nucleotide RNA + 130 kd protein) remove nucleotides from the 5’ end of the rRNA precursors and CCA-adding enzyme adds nucleotides to 3’ end of tRNA; 30% of nucleotides in tRNA are modified In prokaryotes mRNA is NOT further processes. Transcript is DIRECTLY translated. In eukaryotes: mRNA: 5’ capping: 7-methylguanylated-5ppp-5’ cap 3’ poly adenylation: Add ≤250 adenylates, AAUAAA polyadenylation signal CPSF: cleavage & polyadenylation specificity factor bind endonuclease and polyA polymerase adds AAAAAs Splicing: takes place on spliceosome, complex of ≥100 proteins & 5 snRNAs (associate with proteins to give snRNPs) Vertebrate splice site: exon-GU………A…….AG-exon U1 at 5’ splice site, U2 at branch point, U5 at 3’ splice site + U4/U6 !! U6 snRNA and U2 sn RNA form catalytic center of spliceosome!, U4 masks U6 until splice sites are aligned Yields lariat and spliced exons Alternative splicing: membrane vs soluble antibody CTD of RNA Pol II recruits enzymes to synthesis 5’ cap, splicing complex, and endonuclease for poly A tail generation RNA editing: apolipoprotein B in liver vs small intestine; post transcription modification of C to U introduces a stop codon Ribozymes: self splicing RNA, some introns (group 1 introns) The Genetic Code; Mechanism of Protein Synthesis Genetic code: 3 base code, sequential, non-overlapping, degenerate 2 letter: 42 = 16; 3 letter 43 = 64; 4 letter 44 = 254 Adapter molecular hypothesis: Crick, 1958 Codon, reading frame, AUG: start codon; stop codons (UGA, UAG, UAA) tRNA: anticodon, 3’ end is where amino acid is added, 30% nucleotides in tRNA are modified wobble in base-pairing of 5’ anticodon (== 3’ in codon in mRNA) ALL 3’ends of tRNAs end with 5’-…….CCA-3’ aatRNA = activated tRNA aa + ATP = AMP=aa + PiPi aa-AMP + tRNA = aa-tRNA + AMP Aminoacyl tRNA synthetase catalyzes reaction Some Aminoacyl tRNA synthetases can edit wrong aa: Threonyl-tRNA has CCA are that can swing and remove Ser if incorrectly inserted Ribosomes = RNA + proteins Translation: mRNA read 5’ to 3’; protein synthesized in N to C terminus direction Prokaryotic ribosomes: 30S = 21 proteins + 16S rRNA; 50s = 31 proteins + 23S rRNA + 5S rRNA; 30s+50s=70s Eukaryotic ribosomes: 40S = 30 proteins + 18S rRNA; 60 S = 40 proteins + 28S rRNA + 5.8S rRNA + 5S rRNA 40S+60S = 80S Ribosome: three sites – A site and P site and E site RNA is catalyst in ribosome!! Peptidyltransferase activity in 23S rRNA of 30S subunit! Polysomes A site for all aa-tRNA except initiator tRNA P site for all peptidyl tRNA except also for initiator tRNA Eukarytote: first AUG is start codon Prokaryote: first AUG downstream of Shine-Dalgarno sequence (purine rich region) is start site 1st AUG recognized by initiator tRNA (in prokaryote this tRNA carries formylMet), in eukaryotes this is Met 3’ end of 16S rRNA in 30S ribosome subunit binds to Shine-Dalgarno sequence 30S + IF1+IF3+IF2GTP +fMet-tRNA binds mRNA THEN 50S binds, IF1, 3, 2 released and 70S ribosome with mRNA and initiator tRNA aligned at correct position is assembled. EF-TU-GTP brings in each aatRNA to ribosome in A site, using GTP EF-TU-GDP interacts with EF-Ts to lose GDP and bind GTP for next escort function Α-NH2 group of aa makes nucleophilic attack on carbonyl C of ester linking aa to tRNA this transfers growing chains from tRNA in P site to tRNA in A site Peptidyl tRNA in A sites moves to P site via aid of EF-G (translocation) costs GTP Termination: RF1 (UAA & UAG), RF2 (UAA & UGA), RF3 (binds GTP) causes stalling of ribosome Cleavage of 4 phosphoanhydride bounds: ATP→AMP for aa tRNA production (note: ppI → 2 Pi) GTP in elongation (EF-TU –GTP) GTP in translocation (EF-G-GTP) mRNA in eukaryotes is circular because of interactions between proteins that bind the 5’ cap and those that bind the poly A tail The following were not discussed in detail in class in 2015, and you are not responsible for these on Exam #3 in 2015. None-the-less, the concepts are important. Post translational modification Co-translational & post: deformylation of formyl Met, removal of N terminal Met, disulfide bonds, cleavage of proteins, phosphorylation, glycosylation, acetylation Eukaryotes: Signal hypothesis: N-terminal 16-30 aa residues, highly hydrophobic Translation of signal peptide → N-terminus binds to SRP (signal recognition particle) = protein/RNA complex (7 SL RNA + 4 proteins) Binds to SRP receptor (docking protein) on ER Anchored by ribophorins (ribose-binding protein) Signal peptide cleaved by signal peptidase Glycosylation: start in ER finish in Golgi