2/17-2/19 Notes Bios 312
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This 7 page Class Notes was uploaded by Cara Cahalan on Thursday April 7, 2016. The Class Notes belongs to Bios 312 at University of Nebraska Lincoln taught by Karrie Weber in Spring 2016. Since its upload, it has received 11 views. For similar materials see Microbiology in Biology at University of Nebraska Lincoln.
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Date Created: 04/07/16
2/17: Transcription and Translation Readings: 4.74.14 4.7 Transcription Transcription synthesis of RNA from DNA template, copies small units of DNA unlike replication o Transcribe different genes at different frequencies based on the cell’s needs RNA is single stranded with complementary base pairing secondary structure (folding), function depends on 3D shape RNA pol catalyzes transcription, doesn’t require a primer Ribonucleotides are added to 3’OH of the ribose (5’ 3’) RNA pol subunits combine to active enzyme RNA pol holoenzyme o Core enzymeα 2βω, alone synthesizes RNA elongation o Sigma factor σ, not tightly bound, recognize initiation site on DNA and conserved regions Possible to control which genes are expressed by presence/absence of sigma factors Promoters portion of DNA recognized by sigma factor of RNA pol to initiate transcription o Upstream of transcription start site conserved regions Prinbow box 10 region, TATAAT 35 bases, TTGACA Termination GC rich sequence with inverted repeat with central nonrepeating segment o RNA forms stem loop structure followed by run of A transcription terminates o RNA pol pauses at stem loop and dissociated from DNA Rho dependent termination RHO is tightly bound to RNA and moves down chain towards RNA polDNA complex RNA pol pauses at Rhodependent termination site Rho causes RNA/RNA pol release from DNA 4.8 Unit of Transcription mRNA have short halflives due to lack of secondary structure degraded by endonucleases o Rapid turnover allows for quick adaptation to environment tRNA and rRNA have longer due to folding Polytrinsic mRNA mRNA encoding a ground of cotranscribed genes, multiple open reading frames o Several polypeptides are synthesized sequentially by same ribosome 4.9 Transcription in Archaea and Eukarya No operons in Eukarya Promoters 68 base pair “TATA” box 1827 nucleotides upstream of transcription start site o Recognized by TATAbinding protein (TBP) o Upstream of TATA is BRE (B recognition element) that recognized by TFB (transcription factor B) o When TBP is bound to TATA and TFB bound to BRE RNA pol binds transcription RNA processing altering RNA molecule o Exons coding segments. Introns noncoding regions o Introns are removed and exons are joined by splicing (in Eukaryotes) by the spliceosome 2 unique steps of RNA processing in Eukarya o Capping prior to splicing, addition of methylated G at 5’, in reverse orientation initiate translation o Poly(A) tail 100200 adenylate residues, stabilizes mRNA, must be removed to degrade IV: Protein Synthesis 4.10 Polypeptides, Amino Acids, and Peptide Bonds Amino acids contain amine (NH 2 and carboxylic acid (COOH) and unique side chain (R) Peptide bonds link between carboxyl carbon of one amino acid and amino N of other 2 ends of polypeptide: C terminus (free carboxylic acid) and N terminus (free amino group) 4.11 Translation and Genetic Code Translation mRNA protein Genetic code RNA triplet (codon) each with specific amino acid. Anticodon complementary RNA triplet o Degenerate code not oneone recognition (amino acids are not specific to codon, codon is specific) o Wobble only first two position of the codon are specific, third base pair can be irregular Codon bias some codons are preferred over others, rarely used codons will be in small concentrations Open reading frames (OFRs) start codon (AUG), number of codons, stop codon (UAA, UAG, UGA) o Nonsense codons interrupt sense of growing polypeptides, doesn’t add amino acid AUG start codon, adds Nformylmethionine, in middle of codon AUG methionine 0 frame reading frame that translates correct polypeptide (+1 and 1 both create incorrect reading frame) ShineDalgarno sequence ensures proper reading frame, 39 nucleotides preceding initiation codon 4.12 Transfer RNA AminoacyltRNA synthetases binds both amino acid and tRNA possessing corresponding anticodons, ensure each tRNA receives correct amino acid because it recognizes both tRNA carries anticodon, contain some purine and pyrimidine bases that are chemically modified. o Cloverleaf fashion 3’ (acceptor stem) are three unpaired nucleotides (CCA) not encoded in tRNA gene. Nucleotides added by CCAadding enzyme amino acid attached to terminal A of CCA Amino acid + ATP aminoacylAMP + PP aminoacylAMP + tRNA aminoacyltRNA + AMP o AminoacylAMP remains bound to tRNA synthetases until it collides with appropriate tRNA o 2 energy rich phosphate bonds needed to charge tRNA 4.12 Protein Synthesis 30S and 50S 70S ribosome, subunits alternately associate/dissociate and also interact with other proteins Steps of protein synthesis: o Initiation 70S subunit + GTP. 39 nucleotides (ribosomebinding site) helps bind mRNA to ribosome Base pairing between molecules hold ribosomemRNA together securely Polytrinsic mRNA each have RBS translation of several genes on same mRNA AUG (Nterminal has methionine) o Elongation Acceptor (A) site incoming tRNA attaches here, EFTu helps load tRNA Peptide (P) site growing polypeptide chain is help by previous tRNA AUG starts out in P site, new tRNA added to A site translocated to P site open A site Each translocation requires EFG (elongation factor) and one GTP Empty tRNA Exit (E) site Polysome several ribosomes translating single mRNA simultaneously o Termination Stop codon initiates termination. Release factors (RFs) recognize stop codon and cleaves polypeptide releasing finished product ribosomes dissociate Ribosome catalyzes peptide bond formation (23S rRNA) No stop codon ribosome is trapped tmRNA frees stalled ribosome o tmRNA binds defective mRNA and adds alanine and translates short message, contains stop codon release factors bind ribosome disassembles protein is degraded 4.14 Protein Folding and Secretion Secondary structure α helix (H bonding stabilizes) and β pleated sheets Tertiary structure overall 3D shape Quaternary structure number and type of polypeptides in the final protein Denaturation unfolding of protein, retains primary structure so refolding may be possible Chaperonins assist in proper folding of proteins, prevent improper aggregation, refold denatured protein o DNAK/J prevent folding too quickly o GroEL/S fold protein properly Translocases assist in transporting proteins out of the cell Signal sequence beginning (N terminus) of protein molecule, signals cell’s secretory system that this protein needs to be exported, prevents protein from completely folding Lecture: Each gene transcribed individually in Eukaryotes Prokaryotes do not have a nuclear membrane so mRNA can be translated immediately Steps of transcription: o Initiation RNA pol holoenzyme scans DNA for promoter Binding to promoter sequence forms closed complex RNA pol unwinds DNA and begins transcription, sigma factor leaves o Elongation RNA pol continues to move along template, RNA growth continues to termination site o Termination Prevents over expression of genes that are not necessary When complete, RNA pol and RNA is released Inverted repeats result in RNA forming stemloop structures (intrinsic, Rhoindependent) Rhodependent Rho recognize GCrich regions with no secondary structure RNA wraps around Rho pulling towards RNA pol contact between Rho and RNA pol cause termination Antibiotics affecting transcription: Rifamycin B (produced by a bacterium Amycolaptopsis mediterranei) o Selectively targets bacterial RNA polymerase o Binds to the binds to the Mg active site that blocks the exit channel o A synthetic derivative (rifampin) treats tuberculosis, leprosy, contact individuals with bacterial meningitis Steps of translation: o Initiation Initiator tRNA enters at P site o Elongation tRNA guided to A site, peptide bond forms o Translocation/Termination Amino acid shifts from A P site Uncharged tRNA in P site E site and ejected, A is empty to receive next Stop codon enter A site no tRNA binds release factors enter protein released Antibiotics affecting translation: Streptomycin (produced by a bacterium Streptomyces sp.) o Binds to 16S rRNA that forms part of the decoding A site and another protein responsible for codon anticodon binding o At high concentrations binds the 30SmRNAtRNA initiation complex and prevents binding of the 50S 2/19 Metabolic Regulation Readings: 7.17.6 Start Slide 8 of Regulation I I: Overview of Regulation 7.1 Major Modes of Regulation 2 approaches to protein regulation: o Activity of a preformed enzyme, only regulated after synthesis, posttranslational regulation, rapid o Amount of enzyme or protein, regulate level of transcription and translation, slower (minutes) Amount of protein regulated by level of transcription, varying amount of mRNA and then by translation (or not) mRNA Reporter genes encode protein product that is easy to detect and fused to other regulatory elements to monitor gene expression o Green fluorescence protein (GFP) level of fluorescence= level of gene expression II: DNABinding Proteins and Transcriptional Regulation 7.2 DNABinding Proteins Regulatory proteins small molecules interact with, bind to specific sites on DNA turning transcription on/off Major groove main site of protein binding due to size High specificity binding protein must interact simultaneously with several nucleotides Domain region of protein with a specific structure/function Helixturnhelix o Recognition helix interacts with DNA Stabilizing helix stabilizes first helix o Turn three amino acid residues H bonding between recognition helix and specific chemical groups DNA binding proteins can be enzymes that catalyze reactions, binding event blocks(negative) or activates (positive regulation) 7.3 Negative Control: Repression and Induction Negative control prevents transcription Repression sufficient amount of substrate, cell doesn’t produce enzyme, anabolic o Final product of biosynthetic pathway represses enzymes of the pathway o Lactose absent enzymes not produced. Lactose added enzymes are made Induction opposite of repression, enzyme made only when substrate is present, catabolic Effectors: inducer induces enzyme synthesis. Corepressor repress enzyme synthesis o Indirectly bind to specific DNAbinding proteins affect transcription Repressor protein allosteric (conformation is changed when effector molecule binds to it) o Binding to effector repressor protein activated bind operator o Repressor binds operator transcription physically blocked o Repressor protein active in absence of inducer blocking transcription add inducer inactivated repressor protein transcription Operon cluster of consecutive genes whose expression is under control of a single operator, single mRNA o Operator is downstream of promoter Repressor mechanism: prevention of mRNA synthesis by activity of specific repressor proteins that are themselves under control of specific small effector molecules 7.4 Positive Control: Activation Positive control activator activates binding of RNA pol to DNA Transcription requires the binding of an activator protein (maltose) o Maltose activator protein can’t bind to DNA unless first binds maltose (inducer) o Operator known as activating binding site Activator protein helps RNA pol recognize the promoter and begin transcription Regulon more than one operon under control of single regulatory protein 7.5 Global Control and the lac Operon Regulatory mechanism: respond to environmental signals by regulating expression of many different genes global control Glucose is always the preferred substrate!! Catabolite repression controls use of carbon sources if more than one is present o Favored source better C and energy source than other C source, use best first o Indirect result glucose is better energy source. Direct low level cAMP Diauxic growth two exponential growth phases. Better source first grows depleted lag other source Glucose present lac operon is not expressed, lactose is not used. Glucose depleted lac operon activated Activator protein cyclic AMP receptor protein (CRP) o CRP binds to DNA only if it has first bound a small molecule cAMP Cyclic AMPsignaling molecule, synthesized from ATP, glucose inhibits synthesis and stimulated transport of cAMP out of the cell o Glucose high cAMP low in cell For lac operon to be transcribed: if met lac operon transcribed o Level of cAMP must be high enough for CRP proteins to bind CRPbinding site o Lactose present so lactose repressor doesn’t block transcription Lecture: Gene expression: transcription of gene into mRNA followed by translation of mRNA into a protein Constitutive proteins needed at the same level all the time o Microbial genomes encode many more proteins than are present at any one time Regulation is important in all cells and helps conserve energy and resources Inhibition of enzyme activity o End product binds to enzyme inhibition (substrate cannot bind, enzyme reaction inhibited) o No binding enzyme reaction proceeds RNA polymerase must recognize and bind to the promoter in order to transcribe the gene into mRNA o Proteins bind to DNA and either promote or inhibit transcription DNA Binding Proteins: regulation of transcription o Homodimer: two identical polypeptides, i.e. DNA binding proteins o Inverted repeats: each dimer recognizes molecular contacts of each sequence Specificity from amino acid side chains of the proteins and specific chemical groups on the bases DNA binding proteins interact with inverted repeats. (not stem loop structures) Repression: Arginine Operon o Repressor regulatory protein that binds to specific sites on the DNA to regulate transcription o Corepressorrequired for a conformational change and binding of the repressor to the operator o When arginine is added to culture medium—no further synthesis of enzymes required for biosynthesis. Induction: lac operon o Repressor binds transcription blocked o Inducer binds to repressor induce conformational change repressor doesn’t bind transcription Positive: Maltose operon o Activator binding site Promoter sequence weakly binds RNA polymerase (poor match) o Maltose activator protein requires inducer to bind to activator no transcription o Inducer (maltose) binds to activator protein transcription proceeds Key concepts: o Negative control involves a repressor protein that prevents transcription. o Positive control involves an activator protein to facilitate binding of the RNA polymerase to the promoter. o In response to a change in environmental conditions gene expression may also be under global control.
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