RNA znuA or ftsZ Protein ZnuA or FtsZ Watch out for italics not Lecture 11 Microbial Genetics Genus species G species Can refer to abbreviation in document once written fully out first DNA znuA or ftsZ Chromosome main genetic element contains essential genes Gene specific genetic element within the chromosome that encodes the information to make a specific RNA or protein Genome entire complement of genes in cell includes chromosomes and plasmids Most prokaryotic genomes are comprised of a single circular dsDNA chromosome Human genome 3 000 000 000 nt Largest bacteria 13 033 779 nt Smallest bacteria 112 091 nt Plasmid genetic element contains genes but is non essential Replicate separately from chromosome double stranded Genetically mobile circular encode beneficial genes for cell like antibiotic resistance They are expendable Does not have to be transferred Can integrate to chromosome Frederick Griffith s experiment 28 Streptococcus pneumoniae pneumonia G Facultative anaerobe human path Naturally competent uptake DNA from env and insert into own chromosome Smooth Strain Has a capsule causes disease Rough strain No capsule non virulent no disease Demonstrated cells could transform not sure how Rough Strain mouse live Smooth strain mouse die Heat killed smooth strain mouse live Rough Strain and heat killed smooth strain mouse die Avery MacLeod McCarty 44 Systematically depleted sample of ONE component of smooth killed cells to assess rough cell transformation removed RNA Protein Lipids DNA in different test Only when DNA was killed cells didn t turn bad Demonstrated DNA was most likely transforming molecule but did not show directly Streptococcus pneumoniae Hershey Chase Experiment 52 Directly showed that DNA was the transformation molecule Viruses bacteriophage specifically infect bacteria has a protein coat and DNA genome bind to surface of a bacterial cell and inject genetic material Tested whether DNA or Protein was the genetic information that could transform bacteria Radiolabeled Phage Proteins with Sulfur Radiolabeled DNA with Phosphorus Used T2 Bacteriophage and E coli When introduced to a cell the Phosphorus showed up meaning DNA was transferred Meselson and Stahl proved DNA replicates semi conservatively Used E coli N15 mixed with N14 After first cycle 100 14 15 After second cycle 50 14 15 50 14 14 IF CONSERVATIVE 50 15 15 and 50 14 14 after first cycle never mix Semi conservative Lecture 12 Replication Transcription and Translation DNA replication DNA transcription RNA translation Protein Supercoiling DNA coiled to fit compact space of the cell E coli is 800x longer than cell Sometimes hides binding sites Chromosome double stranded most circular 1 per cell bi directional replication Replication starts at origin of replication Goes in both directions Two chromosomes separate termination site Theta Replication peels off looks like a Replication Fork has replisome on it E coli can divide double every 20 mins can start a new round of replication before the first is even complete Rolling Circle replication usually with plasmid replication Nuclease makes a cut yielding a 3 OH group and 5 P group Nucleotides are added to the 3 OH group displacement the 5 P terminated strand Elongation of 3 end continues Transcription Promoter DNA seq reg Exp of gene on off RNA polym binding site Repressor Activator binding sites Coding Strand DNA seq that is identical to the transcribed RNA T in DNA replaced by U in RNA oriented 5 to 3 Non Coding Template Strand Reverse 3 to 5 complement of coding strand Actually read by RNA Polymerase Operon set of different genes transcribed from a single promoter Allows genes to be exp t same time and similar lvls Promoter sits on CODING STRAND RNA Polymerase Enzyme is a large multi subunit enzyme alpha beta beta prime synth RNA core Sigma Region recognizes promoter region initiates transcription Transcription Initiation 1 RNA Polymerase binds to 10 35 region of promoter Sigma factor recognizes sequence 2 RNA Polymerase starts moving down the DNA 5 to 3 3 RNA polymerase starts synthesizing new mRNA at 1 Site 1 is start of transcription first nucleotide transcribed to RNA Consensus sequence most represented base at a specific position Promoter sequence runs from 35 to 10 LoGo Sig70 RpoD TTGACA For most genes major sigma factor for normal growth Sig54 RpoN TTGGCACA Nitrogen assimilation Sig38 RpoS CCGGCG Stationary phase oxidative osmotic stress Sig32 RpoH TNTCNCCTTGAA Heat shock response Sig28 TAAA Flagella synthesis Sig24 RpoE GAACTT Response to misfolded proteins in periplasm Sig19 FecI AAGGAAAAT For iron transport RNA synthesized in 5 to 3 direction like DNA Coding Strand Identical to RNA T U Non coding Template Complementary to RNA T U Actually read Coding 5 GTAGATGCG 3 NonCo 3 CATCTACGC 5 RNA 5 GUAGAUGCG 3 Coding Sequence DNA template for producing RNA transcript Terminator DNA sequence that tells RNA polymerase to stop transcription Rho Dependent Termination Rho is on strand going into polymerase Rho opens up then clamps around a C rich site on the mRNA and begins pulling the mRNA through its central pore When Rho catches up to paused polymerase it continues to pull mRNA separating it from the template and the polymerase Rho Independent Termination Stem loop causes the RNA polymerase to pause A U base pairs are weak after a few second the mRNA will dissociate and the polymerase will release the DNA 1 Start of RNA transcript First nucleotide on RNA RBS Ribosomal binding site AKA Shine Dalgarno Sequence This is where ribosome binds to initiate translation AUG Start codon Tells ribosome to start translating First amino acid in the polypeptide UGA Stop codon The codon that tells the ribosome to stop translating Terminator sequence in the RNA that tells RNA polymerase to stop transcribing Translation synthesis of proteins from mRNA Ribosome protein RNA complex where amino acid subunits are linked Genetic code a triplet of nucleic acid bases codon that encodes a specific single amino acid Specific codons for starting and stopping Anticodon on tRNA recognizes codon on mRNA tRNA is the amino acid w RNA Start AUG Stop UAA UAG UGA Reading frame triplet code that determines what amino acid added to polypep chain Shine Dalgarno Seq AKA RBS aligns ribosome with AUG 16S rRNA contain anti Shine Dalgarno Site Thousands of ribosomes per cell two subunits 30S and 50S in proks 70S complex Combination of rRNA and protein Translation uses tRNA GTP used during initiation
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