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BIOL 205 Lecture Notes

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BIOL 205
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Bendena Lecture Notes - Griffith work on Streptococcus pneumonia which is lethal in mice o Mutant R strain with no polysaccharide coat is non-virulent o Mice injected with heat killed virulent cells and live R cells died  Virulent cells could be recovered from the mice - Destroyed various components of the cell to determine genetic material o Cells treated with DNase; mouse lived; no S strain recovered - Hershey and Chase demonstrated genetic material of phage is DNA; not protein 32 35 o Incorporate35P into DNA and S in protei32  S found in phage ghosts; P found inside bacteria  Phage protein never entered the bacterial cell - DNA has 3 basic components: 1) Deoxyribose 2) Phosphate 3) Nitrogen bases o Purine – double ring structure in adenine and guanine o Pyrimidine – single ring structure; cytosine and thymine - P group attaches at 5’ aspect of the sugar; N base at the 1’ - Chargraff’s Rule  T+C = A+G; A=T, G=C - Nucleotide strands held together by hydrogen bonds - Backbone formed by phosphodiester linkages (phosphate-deoxy alternate)  5’ to 3’ linkage  Two backbones are antiparallel (opposite 5’ to 3’) o Double helixes stack which adds to stability by excluding water  Double helix has major and minor groove  DNA-protein associations are in the major groove o Purine+Purine = DNA thick; Pyrimidine+Pyrimidine = DNA thin - G+C has 3H bonds; A+T has 2H bonds; higher melt point for G+C - Each single DNA strand acts as a template for synthesis - Semiconservative – one strand from original and one strand from newly synthesized DNA - Conservative – parent DNA molecule is conserved; totally new double strand - Dispersive – contain segments of both parental and new DNA o Meselson-Stahl proved that DNA copied semi-conservatively - DNA polymerase adds deoxyribonucleotides to 3’ end of growing strand - DNA pol III catalyzes synthesis at the replication fork (zone of unwinding) o Leading strand – smooth synthesis in the direction of the fork o Lagging – wrong direction of the unwinding (away from rep fork)  Must be in short segments: Okazaki Fragments o Must be initiated by a primer: short chain of nucleotides o Primers are synthesized by proteins: primosome o Primase (RNA polymerase) synthesizes 8-12 nucleotide sequence - DNA Pol I removes the RNA segments and fills them with DNA - DNA Ligase joins 3’ to 5’ ends of Okazaki Fragments - DNA pol 1 and pol III both have exonuclease activity (proofreading) o RNA primer degraded by 5’ to 3’ exonuclease of pol 1 - DNA pol III is part of pol III holoenzyme complex o Two catalytic core and accessory proteins o Beta clamp encircles DNA like donut and keeps pol III attached o Pol III goes from distributive enzyme (adding a few nucleotides), to processive enzyme (adding thousands of nucleotides) o Primase does not touch the clamp (distributive) - Unwinding is done by helicases and topoisomerases o Helicases disrupt H bonds of double helix into single strands o Stabilized by single-strand binding proteins (SSB) - Supercoiling is created or relaxed by topoisomerases o Fig 7-19 p283  DNA Gyrase a topoisomerase - replisome in eukaryotes is more complex; nucleosomes need to be disassembled - Proliferating Cell Nuclear Antigen (PCNA) is the eukary version of beta clamp - Thousands of replication forks in eukaryotic cells; 1 in prokaryotic o Replication in both directions from multiple origin points o Most origins have the same 100-200 bp motifs - Origin of replication complex (ORC) begins replication - Telomeres – cause problems in the lagging strand as there will be a gap o if this were replicated; DNA would continually shorten in subsequent daughter cells o Telomerase – adds short repeats to 3’ ends of DNA molecules  Fig 7-26 p289  Telomerase function and action - Telomeres associate with proteins to form protective caps o Sequester 3’ overhangs would otherwise be seen as double strand breaks - Somatic cells produce little telomerase; experience telomere shortening o Associated with aging - Higher eukaryotes DNA encoded in sequences through introns and exons o Exons – encode parts of proteins (expressed region) o Introns – separate exons (intervening region) - Spliceosome – removes the introns and joins the exons o Process is called RNA splicing; makes mature RNA with continuous protein coding information - The functioning of DNA and RNA is based on two principles o Complementarity of bases responsible for determining of new DNA strand o Certain proteins recognize certain base sequences in DNA - RNA having a rapid turnover was discovered with Pulse-Chase Experiment o Infected bacteria pulsed with radioactive uracil o Any RNA made readily detected with radioactive uracil - 1) RNA usually single stranded nucleotide chain (more flexible) - 2) RNA has ribose sugar (2’ OH group) - 3) RNA contains pyrimidine uracil to pair with A o U can pair with G in RNA folding; not during transcription - 4) Like proteins, but unlike DNA; can catalyze reactions - Messenger RNAs – encode info necessary to make polypeptide chains o Intermediary that passes info from DNA to protein - Functional RNAs – does not encode info to make protein o RNA is the final product; functional as RNA - Two classes of functional RNA found in eukary and prokary o tRNA – bring the correct amino acid to the mRNA o rRNA – major components of ribosomes - Other functional RNAs are specific to eukaryotes o snRNA – unite with protein subunits to form the spliceosome o miRNA – regulating the amount of protein produced o siRNA – inhibit the prod of viruses; - Transcription – production of RNA from copies of DNA nucleotide sequence o RNA produced is referred to as transcript - One of separated strands acts as Template o Only one strand used for every gene; same strand in each gene - Each ribonucleotide positioned opposite it complementary base by RNA pol o Links aligned ribonucleotides to grow RNA molecule o Nucleotides are always added at the 3’ growing end - Nucleotide sequence in coding strand is same as RNA - Transcription has three stages: initiation, elongation, termination o The mechanisms are different in E and P Initiation in P - Promoter – binding site for RNA pol to begin transcription o Also referred to as the 5’ regulatory region o Upstream – occurs before the referred to sequence o Downstream – after the referred to sequence; in transcription direction o First DNA base transcribed is +1; initiation site  Downstream of this (+); upstream (-) - Consensus Sequences – sequence in agreement with most individuals o Similar sequences found at -35 and -10  Note 5’UTR and 3’ UTR from terminat′on and initiation - RNA polymerase holoenzyme has 2𝛼 units, 𝛽,𝛽 ,𝜔 and 𝜎 subunit  𝛼 assembles enzyme, 𝛽 catalysis, 𝛽′ binds DNA  𝜎 binds -35 and -10 regions; position holoenzyme for initiation  Fig 8-8 p 303  Holoenzyme mechanism Elongation in P - Holoenzyme maintains ssDNA region: transcription bubble - Addition of nucleotide from splitting high energy triphosphate (release diP) o 8-9 nucleotides added make RNA-DNA hybrid inside bubble o 5’ end extrudes from holoenzyme as ssRNA Termination in P - Continues beyond the encoding segment of a gene (3’ UTR) - There’s intrinsic and rho dependent termination o Intrinsic mechanism in direct  GC rich segment followed by string of A’s  GC can H bond (3); creating hairpin loop o Rho dependent requires a protein called rho factor  Recognizes termination signals for RNA pol  40-60 nucleotides rich in C residues  Rho facilitates release of RNA from RNA pol - Transcription more complex in E for three main reasons o 1) Many more genes to be transcribed/recognized in E  low gene density so more complex (lots of introns) o 2) E has a nucleus (transcript/translation spatially separate)  RNA modified when leaves nucleus: RNA processing  mRNA is fully processed; pre-mRNA newly synthesized o 3) Template for transcription is organized in chromatin  blocks access of RNA polymerase Initiation in E - GTF’s recognize and bind to sequences in the promoter o GTFs are TFIIA, TFIIB o GTFs and RNA pol II make the pre-initiation complex (PIC) - Sequence TATA seen -30 from initiator site: TATA Box o When transcription is initated most of the GTFs disassociate - 𝛽 subunit of RNA pol II contains carboxyl tail domain (CTD); a protein o Located near the site where new RNA emerges o Elongation begins when CTD is phosphorylated by the GTFs o Weakens the connection of RNA pol II RNA Processing - In P translation begins at 5’ end of RNA while 3’ end is still being made o In E more processing is required before translation - 1) Add 5’ cap 2) splicing introns 3) add 3’ tail of A (polyadenylation) - Processing takes place during RNA synthesis: co-transcriptional - 5’ cap added by several proteins that interact with CTD  Cap is 7-methylguanosine o Protects RNA from degradation; required for mRNA translation - 150-200 A nucleotides added to 3’ cut end o Referred to as poly(A) tail o AAUAAA is the polyadenylation signal - Introns are removed from pre-mRNA while RNA is still being transcribed o Before the transcript is taken into the cytoplasm o Process is called splicing o Brings together the coding exons - >100,000 proteins in humans but only ~25,000 genes o Genes can encode info for more than one protein - Alternative Splicing – different mRNAs produced from same primary transcript by splicing together diff exon combinations  Fig 8-14 p309  Alternative Splicing Small Nuclear RNAs (snRNAs) - Junctions are sites at which splicing reactions take place - Splice junctions were specific nucleotides found to be near identical across genes o Almost always have 5’ GU and AG 3’  GU-AG Rule - snRNA complimentary to the splice junctions  p 310; p311 w Fig 8-16; 8-17 - Protozoan Tetrahymena can excise 413 nucleotide intron from itself without adding any protein o Self-splicing introns o First time things other than proteins were found to catalyze reaction - RNA world – RNA must have been the genetic material in first cells  RNA only material to code genetic info and catalyze reactions Small Interfering RNAs (siRNAs) - Selective shutting off of a gene: gene silencing - Observed in gene silencing by dsRNA, virus resistance in tobacco plants, as well as transgene silencing and co-suppression in a number of plants o Transgene – gene that is introduced into chromo of an organism o Endogenous gene – transgene copied from normal gene in an organism  Normal gene is the endogenous gene o Transgene Silencing – transgene incorporate into chromo but fails to make protein product o Co-suppression – transgene and normal copy fail to produce protein - Transgene silencing and viral resistance through RNA interference (RNAi) o Dicer recognizes long dsRNA molecules; cleaves them into siRNAs o RISC (RNA induced silencing complex) unwinds the siRNAs o Antisense strand still attached to RISC; hybridize with cell mRNAs - Transfer RNA (tRNA) – adapters that translate the codons in mRNA to right AA - Ribosomal RNA (rRNA) – major components of ribosomes; ribos composed of several types of rRNA - Most genes encode mRNA; most cellular RNA is functional RNA o More stable than mRNA (remain in tact longer) o Transcription of functional RNA is most of nuclear transcription - Proteins are the main determinants of biological form and function o Chain of amino acids o Chain is referred to as Polypeptide o R group can be anything from H to complex ring - Peptide bond formed by linking amino end (NH ) to 2arboxyl end (COOH) o Water molecule is removed during peptide bond formation - Primary Structure – linear sequence of amino acids in polypeptide chain - Secondary – local regions of polypeptide chain fold into specific shapes  Most common are alpha helix and pleated sheet - Tertiary – folding of the secondary structure - Quaternary – composed of two or more folded polypeptides (subunits) o Can be btwn diff polypeptides (creates heterodimer) - Protein shape is determined by primary AA sequence and conditions in the cell o Globular proteins – compact structures; enzymes and antibodies o Fibrous Proteins – skin hair and tendons - Domains – AA sequences or conserved protein folds associate with specific functions  Overlapping code Fig 9-5 p325 o Overlapping code predicts single base change will alter up to 3 AA - Genetic code is non-overlapping - Suppressors were used to demonstrate there was a triplet code o Phage T4 can grow on B and K E. coli strains o Mutants in the rII gene can not grow on K strain o Mutants induced by chemical called proflavin - Some reversions were found that could grow on strain K o Second mutation suppressed the original mutation - Addition or deletion of single pair on DNA shifts reading frame on mRNA o Causes all following words to be misread o Insertion after deletion suppresses the deletion; by itself insertion disrupts sequence  P 327 has good example - Combinations
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