Chapter 7 The role of DNA How do we KNOW DNA is the hereditary molecule The process by which cells use hereditary information stored in DNA is very complex DNA must be retained intact yet copied to make new cells DNA replication It must be turned into multiple working copies in the form of RNA to provide instructions to produce enzymes structural proteins transcription The RNA molecules must be read and decoded to form the enzymes structural proteins of the cell translation The systems must have the ability to deal with damage to the core DNA molecules of the cell DNA repair How do we KNOW that DNA is the hereditary molecule The Griffith experiment One of earliest experiments to show that DNA could contribute hereditary information in bacteria was work of Griffith in 1920s with smooth S and rough R strains of Streptococcus pneumonia S strain has a capsule and the organism cannot phagocytize R is not Something he didn t know what at the time could transform nonpathogenic R strain into pathogenic S strain when the two were mixed together Transformation the process of bacteria picking up free DNA from the pathogenic environment How do we KNOW that DNA is the hereditary molecule Avery MacLeod and McCarthy experiment early 1940s to determine whether DNA RNA or protein was responsible for transformation observed in Griffith s experiments Used mixtures from S strains but digested away one component at a time Only the mixture with DNA left over could still transform R cells into S cells indicating DNA was the molecule responsible for transformation They decided that DNA must be the genetic material How do we KNOW that DNA is the hereditary molecule Hershey Chase experiment Some thought bits of protein or RNA could still be present in DNA only mixture of Avery MacLeod McCarty experiments Hershey and Chase used radioactive labeling of either proteins or DNA in bacteriophage A bacteriophage is made of proteins that make up the capsid and DNA inside the protein capsid They let labeled phages infect bacterial cells then determined where radioactivity ended up Only the labeled phage DNA went into the bacterial cells further proving that DNA was the hereditary molecule Structure of DNA Despite evidence on WHAT the hereditary molecule was the STRUCTURE was still not characterized Rosalind Franklin James Watson and Francis Crick early 1950s described the now well known double stranded double helix structure of DNA Each nucleotide building block of DNA consists of five carbon sugar 2 deoxyribose Phosphate group attached to the 5 of the sugar Nitrogenous base attached to the 1 of the sugar One strand of DNA is complementary to the other strand A pairs with T G pairs with C by hydrogen bonding Phosphodiester bonds form the sugar phosphate backbone of each strand overall structure of DNA is same across all three domains but the way it is packaged is not Bacteria A single circular chromosome must be supercoiled to fit into cell Archaea A single circular chromosome packaged around histones Eukarya multiple linear chromosomes packaged around histones As usual there are always exceptions D radiodurans may possess 4 to 10 copies of its genome They are all stacked up on on top of another stacked genome copies may help give this bacterium its strong radiation resistance DNA replication How does DNA replicate DNA replication is semi conservative each time dsDNA is copied each copy has one strand of the original molecule and one newy made strand Origins of replication after structure of DNA was revealed how it was copied took more research 10 years after Watson Crick s work Jacob Brenner and Cuzin discovered how DNA replication was initiated in E coli cells DnaA protein binds to oriC DnaB a helicase is recruited with DnaC a helicase loader DnaG a primase is recruited to lay down initial RNA primers at the origin of replication needed for DNA polymerase to work Single stranded DNA binding proteins help keep the DNA unwound Eukaryal replication initiation Multiple origins of replication on each chromosome Otherwise it would take too long to copy them Chromosomes much larger so need multiple starting points for replication Studied in yeast which are small single celled eukaryotes that have some things in common with bacteria similar to process in bacteria just using different proteins Major enzymes involved in DNA replication in bacteria Helicase unwinds helix at replication fork Single stranded binding protein prevents open helix from annealing Primase primes new strand of DNA at the origin of replication to get things started DNA Polymerase III major polymerizing enzyme DNA Polymerase I excises removes RNA primer and fills gaps with DNA Ligase seals nicks in DNA DNA replication Initiation and elongation Once replication fork forms DNA polymerase III adds nucleotides to 3 OH end of RNA primers DNA is made in the 5 to 3 direction A continuous leading strand and a discontinuous lagging strand forming Okazaki fragments are formed virtually identical in bacteria and eukarya lagging strand can t be left in fragments Different DNA polymerases but they perform the same basic function DNA polymerase I removes RNA primers and fills in gaps with deoxyribonucleotides DNA ligase seals sugar phosphate backbone creates phosphodiester bonds Termination of DNA replication of circular chromosome Tus proteins bind ter sites to stop elongation Transcription How are genes transcribed What is a gene segment of DNA that gets transcribed copied into ssRNA Transcription has specific initiation elongation and termination processes RNA is slightly different from DNA How RNA is single stranded typically DNA is double stranded RNA has a nitrogen based Uracil instead of Thymine RNA is made up of ribonucleotides but the sugar is slightly different RNA has an oxygen Transcription What is a gene There are different forms of RNA and each serves a different purpose in the transcription translation processes of gene expression Messenger RNA mRNA Encodes proteins Average 1000 1500 bases nucleotides nitrogen bases long Transfer RNA tRNA Carries amino acids to the ribosome to build a protein during translation Never translated into a protein Ribosomal RNA rRNA Component of ribosomes site of protein synthesis Never translated into a protein Transcription Initiation and elongation transcription starts at a promoter sequence of DNA know what a promoter is RNA polymerase separates DNA and makes a complementary strand of RNA by reading the DNA