Biochemistry I (CHE 418 / 5418 Reading Assignment Berg et. al (2012) Chapter 4 β.

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Biochemistry I (CHE 418 / 5418 Reading Assignment Berg et. al (2012) Chapter 4 β

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  • Biochemistry I(CHE 418 / 5418Reading AssignmentBerg et. al (2012) Chapter 4

  • Central Dogma of Molecular Biology DNARNA Protein mRNA. tRNA, rRNA, snRNA NucleusReplication - DNA directed DNA synthesisTranscription - DNA directed RNA synthesisProcessing of mRNA capping, polyadenylation, splicingCytoplasmTranslation - RNA directed Protein synthesis Transcription TranslationReplication

  • Functions of Nucleic AcidsBuilding blocks of DNA and RNADNA = Genetic materialRNA = Adapter molecule between DNA and proteinTransport chemical energy within the cellATPSignal molecule cyclic AMP

  • Nucleic AcidsNucleic Acid - linear, non-branched polymer of nucleotides

    Classes of Nucleic AcidsRNA = ribonucleic acidDNA = 2' deoxyribonucleic acid

  • NucleotideNucleotide contains: Pentose sugarNitrogenous basePhosphateOne or more

    Adenosine Triphosphate (ATP)

  • NucleotidePentose sugarCarbons are numbered with primes to differentiate between carbons / nitrogens of nitrogenous bases -D-2-DeoxyriboseDNA OnlyRiboseRNA Only

  • Nucleotidephosphate group

  • Pyrimidine BasesCytosine(C )4-amino-2-oxypyrimidineDNA and RNAThymine(T)5-methyl-2,4-dioxypyrimidineDNA ONLYUracil(U)2,4-dioxypyrimidineRNA ONLYPyrimidine

  • Purine BasesAdenine (A)6 aminopurineDNA and RNAGuanine(G)2- amino-6-oxypurineDNA and RNAPurine

  • Sugar Phosphate BackboneNucleotides connected by 3 to 5 phosphodiester bondImparts uniform negative charge to DNA / RNANegative charge repels nucleophilic species (e.g. hydroxyl) thus the phosphodiester bond resists hydrolytic attack.Separation by agarose gel electrophoresisCreates 3 and 5 end (directionality) Convention: Nucleotide sequences are written 5 to 3, L to R

  • Bases are attached to sugar by Beta Glycosidic linkageN-9 of purine and N-1 of pyrimidine

  • NucleotideNucleoside = sugar + nitrogenous base,

    Nucleotide = sugar + nitrogenous base + phosphate.Adenosine monophosphate(A nucleotide)Adenosine(A nucleoside)

  • What data did Watson and Crick use to determine the structure of DNAX ray diffraction photograph of DNA crystalsChargaffs rulesBond angles from reference booksBuilt models

  • Erwin Chargaffs RulesEdwin Chargaff determined the composition of DNA from many organisms[A] = [T][G] = [C]

  • DNA is a HelixX ray diffraction photographMaurice Wilkins and Rosalind FranklinTwo chains that wind in a regular helical structure.

  • Watson and Crick (Complementary) Base PairingNucleotide content determinesmelting point of DNA.

  • Double HelixB formDiameter of helix = 20.0 (2.00 nm)10.4 base pairs / turn; 34 (3.4 nm)1 base pair 3.4 (0.34 nm)NoteComplementary base pairingMajor groveMinor groveAntiparallel Hydrogen bonding between complementary base pairs.

  • DNA held together by hydrogen bonding and Hydrophobic interactionsHydrogen bonding between base pairs4 21 kJ / mol (1 5 kcal/mol)Hydrophobic interactions (van der Walls) due to base stacking.2 4 kJ / mol (0.5 1.0 kcal / mol)

  • Forms of DNAB formNormal formWatson and Crick formA formdehydrated B formnucleotide tilted 20o relative to helical axisZ form (zig zag)stretches of alternating purine / pyrimidinesbase pairs flip 180o Left handed helix

  • DNA is Organized into GenesGene discrete, functional unit of DNA when expressed, (transcribed) yields a functional product rRNA, tRNA, snRNAmRNA - translated into a polypeptide sequence.Open reading frame - long stretch of nucleotides that can encode polypeptide due to absence of stop codons.

  • Chromatosomes Pack to FormChromatin FibersHistones H1, H2A, H2B, H3, H4Histones contain (>20%) arg and lys ---basic amino acids

  • KaryotypePhotograph of chromosomes from a single organismArranged by size (largest to smallest)Homo sapiens 46 chromosomes23 pairs3 billion base pairs (hapliod)25,000 genes

  • Chromosome ContainsCentromere site that connects sister chromatidsKinetochore attachment site of spindle to chromosomeTelomere - nucleotide repeat at end of linear chromosomeTTAGGG x 1000synthesized by telomerase

  • Properties of DNAMelt / Anneal / ReannealHypochromic effectsSupercoiled / Relaxed

  • dsDNA can Reversibly MeltHeating DNA breaks hydrogen bonding between base pairs.Acid or base also worksTm = melting temperatureHalf the helical structure is lostSingle stranded DNA absorbs light more efficiently than double stranded DNA

  • Hypochromic effect (Hypochromism)DNA can melt and then re-anneal.

    If sequences are similar, they will reanneal or hybridize.

  • DNA exist as Linear or Circular MoleculesProkaryotic, Mitochondrial and Chloroplast genomes are circularCircular molecules may exist in topological isomers RelaxedSupercoiledEukaryotic genomes are linear molecules

  • Single Stranded Nucleic Acids can form complex structuresStem Loops are produced by H-bonding between complementary regions in DNA and RNA.

    Mismatches are observed

  • Hydrogen bonding stabilizes more complex structures.Often observed in ribosomal RNA moleculesSingle Stranded Nucleic Acids can form complex structures

  • Replication"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.Watson, J.D. and Crick, F.H.C., Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid, Nature, 171, pp. 737-738, (1953).

  • DNA may be labeled with 15NGrow E. coli in media containing 15NH4Cl and 14NH4Cl.Purify DNASeparate DNA using density gradient equilibrium sedimentationCsCl gradient from 1.66 1.76 g / cm

  • Testing the Semiconservative Replication HypothesisMatthew Meselson and Franklin Stahl (1958)Grew E. coli in 15NH4Cl until DNA was completely labeled.Transferred E. coli to 14NH4Cl containing media.Followed labeling pattern of DNA through several generations using density gradient equilibrium sedimentation

  • DNA ReplicationDNA directed DNA synthesisDNA Polymerase adds deoxyribonucleotide units to an existing DNA molecule in a template directed fashion in the 5 to 3 direction. E. coli DNA Pol I isolated by Author Kornberg (1958)DNA Polymerase requiresFour dNTPs (dA,T,dG,dC)Divalent cation (Mg2+)Template DNAPrimer provides 3 OH

  • DNA Polymerase Reaction MechanismNucleophilic attack by the 3 OH on the alpha phosphate group of dNTPPPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)

  • Types of RNATypes of RNARibosomal RNA (rRNA) part of the ribosomeTransfer RNA (tRNA) - Messenger RNA (mRNA) sequence translated into protein sequence.Small nuclear RNA (snRNA) involved in splicing (spliceosome)Micro RNA (mi RNA) small RNA complementary to mRNA that inhibits translation of the mRNASmall interfering RNA (siRNA) small RNA that binds to mRNA causing destruction of mRNA

  • TranscriptionDNA directed RNA synthesis RNA Polymeraseadds ribonucleoside triphosphate units to an existing DNA molecule in a template directed fashion in the 5 to 3 direction. RNA Polymerase requires:Four NTPs (A,U,G,C)Divalent cation (Mg2+)Template DNANO primer requiredLacks endo and exo nuclease activityRNA molecules are complementary to the DNA template.

    One prokaryotic RNA PolThree eukaryotic RNA PolRNA Pol IRNA Pol IImRNARNA Pol III

  • Structure of RNAGenes may or may not be transcribed depending on the needs of particular cell type. gene is a functional region of DNAexpressed genes are TURNED ONunexpressed genes are TURNED OFF

  • RNA Polymerase Reaction MechanismNucleophilic attack by the 3 OH on the alpha phosphate group of NTP (ribonucleoside triphosphates)PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)

  • RNA molecules are complementary to the DNA template.mRNA is complementary to template strandmRNA is identical (except for U to T changes) to the coding strand.

  • Prokaryotic Promotor Pribnow box (also called TATA box)5TATAAT 3 centered at -9/-10designated by the 5 to 3 sequence on the NONtemplate strand8 to 10 nucleotides left (5 or upstream) of transcriptional start site (designated +1 --- there is no 0 nucleotde)-35 sequence 5TTGACA3 centered -35 from

  • Eukaryotic Promotor Class II genesthose synthesized by RNA Pol II.Pre mRNA and snRNAPartsTATA or Hogness boxGC box (GGGCGG)CAAT box

  • Transcriptional TerminationRho dependentInvolves protein called Rho Rho independentInvolves stem loop structure in mRNAStem loop is followed by UUUs

  • mRNAProkaryotic mRNA are polycistronicMay encode two or more proteinsEukaryotic mRNA are monocistronicEncode only one protein

  • Eukaryotic mRNA are Posttranscriptionally ModifiedCappingattachment of 7-methylguansine using 5 to 5triphosphate linkagePolyadenylationattachment of 40 to several hundred adenine nucleotides to 3 end of mRNASplicingremoval of introns

  • Amino acids are attached to 3 end of tRNA Aminoacyl-tRNA synthestaseattaches amino acid to tRNA

  • TranslationStages of TranslationInitiationassemble and align ribosome, mRNA, and tRNAfMetElongationtemplate directed synthesis of proteinsTerminationtermination factors halt protein synthesisribosome, mRNA and new protein dissociate Orientation of TranslationRibosomes move 5 to 3 along mRNAProtein is synthesized N to C

  • Genetic CodeMarshall Nirenberg, Har Gobind Khorana, Frances CrickSpecific- Unambiguousspecific codon always codes for SAME amino acidThree nucleotides (codon) = one amino acid61 codons encode amino acidsCodons encoding one amino acid usually differ in the last base.3 codons encode stop codons (UAA, UAG, UGA)Universalconserved from species to speciesmain exception = mitochondriaRedundant (also called degenerate)amino acid may have more than one codonNonoverlapping and comma less (no puctuation)read from fixed starting point (AUG)lacks punctuation between codons

  • Codon Usage TablemRNA are read three nucleotides (codon) at a time starting from a fixed point.

  • Translational Start SiteAUG encodes Met (n-terminal amino acid).Prokaryotes use a Shine-Dalgarno sequence to align a ribosome on the mRNA upstream ( 5) of AUGEukaryotes use the 5Cap to align the ribosome on the mRNA

  • Mitochondrial Genetic Code differs from the Universal Code

  • Eukaryotic mRNA contain Exons and IntronsPhilip Sharp and Richard Roberts (1977)Exons coding regionsIntrons noncoding regionsintervening sequences

  • Introns were discovered by hydridizing mRNA to genomic DNA

  • SplicingRemoval of intronsSpliceosome specific proteins and small nuclear RNA.Most introns start with GU and end with AG

  • WHY UNDERSTAND TRANSLATION?Many Antibiotics kill bacteria by inhibiting prokaryotic translation!!!