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2/17-2/19 Notes

by: Cara Cahalan

2/17-2/19 Notes Bios 312

Cara Cahalan
GPA 3.8

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Notes for Exam 2 including transcription/translation and first lecture on metabolic regulation.
Karrie Weber
Class Notes
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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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