Lecture Week 5 Microbial Genetics PDF

Microbial Genetics Chapter 7 genotype phenotype DNA sequence Physical cha...

Microbial Genetics Chapter 7 genotype phenotype DNA sequence Physical characteristic 1 ATGGGCTCAA AAGTAGCAGG TAAAAAGAAA ACCCAGAATG ATAATAAACT AGATAACGAA 61 AATGGTTCAC AGCAGCGCGA AAATATCAAT ACCAAGACGC TTTTGAAAGG CAACCTTAAG 121 ATATCAAATT ACAAATATCT TGAGGTGATT CAACTAGAAC ATGCTGTGAC AAAATTGGTG 181 GAGTCTTACA ATAAAATAAT TGAACTTTCA CCAAATTTGG TAGCTTACAA TGAAGCCGTT 241 AACAATCAAG ATAGAGTGCC TGTTCAGATA CTTCCTTCTC TATCACGTTA TCAATTAAAA 301 CTGGCAGCTG AATTAAAAAC CTTACATGAT CTTAAAAAAG ACGCAATTTT GACAGAAATT 361 ACTGATTATG AAAATGAATT TGATACTGAA CAAAAGCAAC CTATATTGCA AGAAATCAGT 421 AAAGCTGATA TGGAAAAGTT AGAGAAGCTA GAACAAGTTA AAAGGGAAAA GCGAGAGAAA 481 ATAGATGTAA ATGTGTACGA AAATTTAAAT GAAAAGGAAG ATGAAGAAGA AGACGAAGGA 541 GAAGATAGCT ATGACCCAAC AAAGGCTGGT GATATCGTCA AGGCAACAAA ATGGCCTCCA 601 AAATTACCAG AGATTCAAGA TTTAGCGATT AGGGCCAGGG TATTTATTCA CAAATCCACA 661 ATAAAGGATA AAGTTTACTT GTCTGGATCA GAAATGATCA ACGCACATAA TGAAAGACTA 721 GAATTCCTAG GCGATTCGAT CTTAAATTCT GTTATGACAT TAATTATTTA TAACAAGTTT 781 CCCGATTACA GCGAGGGTCA GTTATCAACA TTAAGGATGA ATTTGGTAAG CAACGAACAG Learning Objectives Week 5 Explain DNA structure and replication. Explain to your friend or me the central dogma of biology. Understand how bacterial and eukaryotic genomes differ.  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Transcription Translation Mutation and transformation Ames test The development of the Ames Test (Not on the test)  Genetics is the study of inheritance (heredity) Transmission of traits from parent to offspring Expression and variation of those traits Structure and function of genetic material How genetic material changes (mutation or transformation) How changes affect organism phenotype DNAEx.sequence Physical characteris fitness or appearance Microbial Genetics: Why should you care? We can use microbial genetics to make bacteria or yeast do what we want. Insulin, Hepatitis B vaccine Heredity is responsible for many human diseases and character traits. Microorganism genetics are much  The Language of Genetics: Gene- a linear sequence of nucleotides that encodes for assembly of specific amino acids into a protein ATGCGCG AT (house) Gene locus- particular location on Bacterial Genome a chromosome for a given gene (address) Genome- all the DNA from a species (neighborhood) Genetic code- the basic language of protein synthesis, nucleotide triplets in DNA, how we get from  Genome size or number of genes does NOT necessarily correlate with organism complexity. Genomes vary greatly in size and number of genes. Smallest viruses-5 genes E. coli- 4,288 genes Roundworm- 19,000 genes Human- 25,000 genes Water Flea- 31,000 genes Corn- 50,000 genes Eukaryotes/Prokaryotes/Viruses all package genetic material differently Figure 9.2 Take home messages (Why microbial genetics) Microbial genetics is easier to study than human genetics Genome size does not correspond to organism complexity. The core of our studies will examine how DNA sequence causes a change in organism phenotype. (The relationship between genotype and phenotype.)  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Transcription Translation Mutation and transformation Ames test The development of the Ames Test (Not on the test)  Prokaryotic (bacteria) genomes are circular and NOT found in a nucleus. Comprised of two structures: Nucleoid Plasmids Nucleoid  Nucleoid are found in prokaryotes (bacteria). Region of cytoplasm where DNA is located Not membrane bound Most bacteria have a single, circular chromosome, few species have 2 or more Smaller circular  Plasmids are small circular DNA molecules Small, circular molecules of DNA that replicate independently (“extra chromosomal DNA molecule”) Carry information required for own replication and often for selective traits Not essential for metabolism, growth, or reproduction Can confer survival  We can genetically manipulate natural plasmids to alter bacteria phenotype. bacteria 1 l a smid Plasmid P bacteria 1 bacteria Plasmid2 Plasmid Pla sm id 2 bacteria 3 Plasmid 3  Natural plasmids can be Fertilityclassified by function. (F-plasmid) - contain tra- genes, capable of conjugation Resistance (R-plasmid) - contain antibiotic and toxin resistant genes; previously known as R- factors Bacteriocin (Col-plasmid) - contain genes that code for production of proteins that can kill other bacteria Take home messages (bacterial genomes) Bacterial genomes are small in comparison to eukaryotic genomes. Bacterial genomes are composed of circular chromosomes and small circular plasmids. Plasmids carry useful genes and can be transferred between bacteria quickly. Bacterial genomes are not in a nucleus.  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Transcription Translation Mutation and transformation Ames test The development of the Ames Test (Not on the test)  Where is Eukaryotic DNA in the cell? Two Classes of DNA: Nuclear DNA - DNA in nucleus (genomic) Extranuclear DNA – DNA outside of nucleus (organelle); mitochondria or chloroplast DNA is comprised of Nucleotides. 5’ 4’ 1’ 3’ 2’ Nucleic Acid Structure (DNA) two lanes of a highway going opposite direction  DNA is a very long strand of Nucleotides. Covalent bonds form sugar- phosphate backbone of each strand Each sugar attaches to two phosphates One bond is to the 5’ carbon The other is to the 3’ carbon Strands are in an anti-parallel arrangement  Nucleic Acid Base Pairing in DNA gen bonds Base Pair the two strands of DNA tog  Key Points about DNA Replication. An anabolic polymerization process that requires monomers and energy Triphosphate deoxyribonucleotides serve as both the monomers and a source of energy One of the keys to DNA replication is complementary structure of the two strands Group questions 1) A pairs with_____. G pairs with ________. Why is this significant? 2) Nucleotides are composed of what three parts? What do each of these parts do? DNA Replication is Semiconservative.  The Process of DNA Replication is complicated and very fast (~1 hour for humans, 3 billion bases)! Requires the actions of 30 different enzymes Separate the strands Copy its template to produce two new daughter molecules  Key Steps of DNA Replication 1. Helicases unwind DNA double helix. 2. DNA polymerase binds to one strand of DNA and begins using it as template for assembling leading strand of nucleotides on 3’ OH- 3. DNA replicates only in the 5’ to 3’ direction; strands are antiparallel so synthesized differently 4. DNA polymerase binds to the other template strand and synthesizes discontinuous segments of nucleotides in the 5’ to 3’ direction (Okazaki fragments) Rules for DNA 5’ and 3’ ends DNA polymerase only extends (makes DNA) in the 5’ to 3’ direction. 5’ end must be across from a 3’ end. DNA replication is different on the leading vs lagging strands Synthesis of the leading strand is continuous. Synthesis of the Lagging Strand is NOT continuous. Rules for DNA 5’ and 3’ ends DNA polymerase only extends (makes DNA) in the 5’ to 3’ direction. 5’ end must be across from a 3’ end.  Bacteria use a single origin of replication as opposed to many and have a circular chromosome. Figure 9.7  Elongation and Termination of Daughter As Strands replication proceeds, newly produced double strand DNA loops down DNA polymerase I removes RNA primers and replaces them with DNA When the forks come full circle and meet, ligases move along the lagging strand – DNA in the human cells is 2 meters long. How does it fit inside the nucleus? Eukaryotic Chromosomal Packaging Take home messages (Eukaryotic Genomes and DNA replication) Eukaryotic genomes are kept in their nucleus and are tightly packaged into chromatin. DNA replication is semiconservative as each new cell gets one old and one new copy of the genome. The leading stand of DNA replication is continuous while the lagging is not continuous. DNA replication moves in the 5’ to 3’ direction.  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Transcription Translation Mutation and transformation Ames test The development of the Ames Test (Not on the test) Central Dogma of Biology!  Transcription is the process of synthesizing RNA using DNA as a template. Transcription takes place in the Nucleoid of prokaryotes Transcription takes place in the nucleus, mitochondria, and chloroplasts of eukaryotes Transcription makes RNA, these RNA molecules have a wide variety of functions! messenger RNA (mRNA): translated into polypeptide ribosomal RNA (rRNA): used in building ribosomes transfer RNA (tRNA): RNA molecules that carry amino acids to growing polypeptide Figure 9.8 The Steps of Transcription 1. Protein transcription factors bind to promoter sites, usually on the 5′ side of the gene (initiation) 2. RNA polymerase binds to transcription factors to open DNA double helix 3. RNA polymerase proceeds down one strand moving in 3′ → 5′ direction 4. As RNA polymerase travels, it assembles nucleotides (elongation) 5. When transcription is complete, transcript is n of Transcription makes sure that transcription happens at the 5’ of genes.  Elongation of the RNA Transcript: Synthesizes the RNA molecule by adding single nucleotides to the 3’ end of the growing RNA molecule.  Elongation of the RNA Transcript: Synthesizes the RNA molecule by adding single nucleotides to the 3’ end of the growing RNA molecule. 5’ 5’ 5’ 5’ 5’ Termination of transcription stops the elongating transcript, can be protein dependent or independent Figure 7.7c In bacteria genes are transcribed as groups called operons. Eukaryotic genes are not assembled into groups Every eukaryotic gene has its own promoter and is independently transcribed. Figure 9.18 t eukaryotic genes do not exist as an uninterrup series of triplets coding for a protein –Introns- sequences of bases that do not code for protein –Exons- coding regions that will be translated into protein Splicing is a very complicated process involving many Proteins and RNA molecules most of which is beyond – A spliceosome recognizes thethe scope of this course. exon-intron junctions and enzymatically cuts them – The exons are joined end to end – Some introns have additional functions aside from separating exons (in humans, introns represent Figure 9.1798% of the All cells in our body have the same genetic code but we have many different cell types, how? Gene expression!  Relationship between genotype and phenotype not always direct Same DNA strand in different individuals may result in different traits because of other DNA or environment DNA strand is expressed only if it is transcribed Must be between promoter and terminator Cells regulate activity of genes by changing rate of transcription DNA may be “silenced” through chemical changes to protein components of chromosomes Once produced, proteins interact with other proteins in cell and combinations of these interactions produce a trait  Bacteria organize collections of genes into operons – Coordinated set of genes regulated as a single unit – Either inducible or repressible Inducible- operon is turned on by substrate the genes act on Repressible- contain genes coding for anabolic enzymes; several genes in a series turned off by product synthesized Control mechanisms ensure that genes are active only when their products are required – Enzymes are produced as needed One of the best understood systems for explaining control of gene expression is the lactose operon (lac). Regulates lactose metabolism in Escherichia coli Three important features: – The regulator (a gene that codes for a protein capable of repressing the operon [a repressor]) – The control locus Promoter- recognized by RNA polymerase Operator- a sequence that acts as an on/off switch for transcription – The structural locus, made up of Take home messages Transcription is the process of making RNA using DNA as a template. Transcription is responsible for making all RNAs. Transcription can be separated into the steps of initiation, elongation, and termination. Eukaryotes genes contain introns and functionally increase the size of the genome without adding actual genetic material.  Group Questions 1) How are transcription different between bacteria and Eukaryotes, how are they the same? 2) Describe the benefits and drawbacks to the operon vs single gene transcription.  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Transcription Translation Mutation and transformation Ames test The development of the Ames Test (Not on the test) Central Dogma of Biology! genotype phenotype Transcription always makes RNA but it makes many different types! messenger RNA (mRNA): translated into polypeptide ribosomal RNA (rRNA): used in building ribosomes transfer RNA (tRNA): RNA molecules that carry amino acids to growing polypeptide Figure 9.8 tRNAs bind to a specific amino acids and add them to a growing protein. Figure 7.12 Figure 9.10 Figure 9.13 Ribosome is a very complex biochemical machine made up of many proteins and RNA molecules. The tRNAs bring amino acids to the rRNA which then links the amino acids together. Figure 9.12  Stages of RNA Translation Three stages: 1. Initiation 2. Elongation 3. Termination All stages require additional protein factors Initiation and elongation require energy (ATP and or GTP) Translation initiation requires mRNA, tRNA, and ribosomal subunits align in a specific orientation. Translation elongation Translation Termination: Amber, Ochre, and Opal codons Release factors recognize stop codons Modify ribosome to activate ribozymes Ribosome dissociates into subunits Polypeptides released at termination may function alone or together Take home messages Translation is the process of synthesizing proteins by translating the genetic code from RNA into a chain of amino acids. Translation can be separated into 3 steps initiation, elongation, and termination. Stop codons are specific codons that do not code from an amino acid and instead terminate translation. Translation requires rRNA (ribosomal RNA) , mRNA (messenger RNA), and tRNA (transfer RNA), as well as many protein factors. Central Dogma of Biology! Change DNA Change in protein and in turn Phenotype  How can we change DNA? 1. Add DNA to an organism. 2. Alter the existing DNA of an organism.  How can an organism add DNA? Genetic alteration of a cell resulting from the uptake and expression of foreign genetic material, DNA (genetic recombination) Via viruses (transduction) Via cell-to-cell contact (conjugation) Free DNA (nonspecific) (transformation) Cells that take up foreign DNA = competent results from alterations in cell wall and cytoplasmic membrane Griffith pioneered these ideas  Griffith’s Experiments on ‘transforming principle’ First experiments suggesting bacteria capable of transferring genetic information through Figure 7.29  Transduction: bacteria accept DNA from virus (bacteriophage) Figure 7.30  Conjugation: Bacteria exchange DNA with another live bacteria Often beneficial to recipient: antibiotic resistance, other xenobiotic tolerance, or new metabolit Figure 7.31 Types of Intermicrobial DNA exchange Group Questions 1) Does transformation of bacteria always lead to a change in phenotype? Why or why not? 2) Imagine you have a change in your DNA sequence. Explain how this would effect transcription. Explain how this could effect translation of this DNA sequence.  Outline for the weeks lecture: Microbial Genetics: what is it, why you should care, and the language of genetics Bacterial genomes Eukaryotic genomes DNA replication Mutation and transformation Ames test The development of the Ames Test (Not on the test) Transcription Translation  A mutation is a heritable change in Mutations can result inDNA the wrong protein synthesized or change where and how much of a protein is made Silent, Missense, and Nonsense Mutations Mutations can be beneficial, harmful, or neutral Although our DNA replicates MILLIONS OF TIMES A DAY – there is a very low rate of mutation (