Microbiology Chapter 8 Microbial Genetics 1 Genetic Material Genetics - the science of heredity, including the study of genes, how information is carried, and how this information is replicated and passed on to subsequent generations Genes - segments of DNA which code for functional products Chromosomes - the physical structures which carry the hereditary information in the cell 2 Physical Structure of DNA Four bases found in DNA: Two purine bases: Adenine (A) Guanine (G) Two pyrimidine bases: Cytosine (C) Thymine (T) [equivalent base in RNA is Uracil (U) 3 Base Pairing Bases associate in DNA by complementary base pairing A always base pairs with T (or U in RNA) G always base pairs with C 4 Information Transfer The DNA (gene) stores the hereditary information, to be used the information must first be transcribed (transcription) into mRNA and then the mRNA is translated into protein 5 Genetic Terminology KEEP IT STRAIGHT – are we talking about the gene or about the protein made from the gene? Naming conventions help… Genotype - the genetic makeup of an organism, the genes which encode particular characteristics of the organism (collection of genes). Determined by actual DNA sequence (gene) written pyrBPhenotype - the actual, expressed properties (observed) of the gene. The result of phenotype is a protein (or collection of proteins) written Pyr- or PyrB- (note use of capitalization when referring to protein) One phenotype can possibly be the result of different genotypes 6 Bacterial Chromosome Bacteria generally have a single, circular chromosome e.g. E. coli chromosome consists of 4 million base pairs (4 x 106 bp) this is a medium sized bacterial chromosome if the average gene is 1000 bp in size, then this chromosome would contain an estimated 4,000 genes 7 DNA Replication (2 major points on this slide) DNA is double-stranded. Replication is the faithful copying of each strand. Since base pairing is specific, each strand can serve as the template for the opposite One parent molecule of double-stranded DNA gives two daughter molecules each daughter molecule contains one "original" strand and one newly synthesized strand 9 this is called semiconservative replication 10 G C G C G C T A T A T A A T A T A T CG C G C G G C G C G C T A T A T A Directionality of DNA Strands Direction is determined by the sugar/phosphate backbone OH end is the 3’ (pronounced three-prime) end P end is the 5’ (five-prime) end. DNA strands are antiparallel One occurs in the ‘opposite direction’ of the other. 11 Replication Fork Replication occurs at a replication fork in most bacteria, there are two forks which are moving in opposite directions (bidirectional replication) 13 notice twin replication forks Activity at Replication fork Synthesis occurs in a specific direction on the DNA. Synthesis ALWAYS occurs 5’ 3’ One strand is the leading strand. This strand runs into the unzipping fork in the 5’-3’ direction, so open space is always in front of the newly forming strand. 15 DNA Polymerase Leading strand DNA Helicase Lagging strand DNA Ligase Replication Fork continued the other is called the lagging strand because it cannot synthesize continuously due to direction of DNA The short pieces on the lagging strand are called Okazaki fragments Lagging strand synthesis requires RNA primers to begin each segment. DNA Polymerase requires a free end to start from. It can’t start at an empty space. DNA Polymerase can’t fit against ends of earlier segments so it leaves a small gap. These gaps are closed by DNA Ligase 17 RNA Three types of RNA are made in the cell, all are involved in protein synthesis Ribosomal (rRNA) is a required part of ribosomes - the machinery of protein synthesis Transfer (tRNA) brings the amino acids to the ribosomes in a specific manner Messenger (mRNA) carries the coded information for the synthesis of specific proteins All RNA is transcribed from DNA by RNA polymerase 20 RNA Transcription RNA polymerase transcribes mRNA using the DNA template (the "coding" strand of the double-stranded DNA) the new RNA strand has ribonucleotides instead of deoxyribonucleotides & uracil (U) is used in place of thymine (T) to base pair with adenine (A) RNA polymerase binds to a promoter (special start site on DNA), then polymerizes the new chain using complementary bases polymerization stops upon reaching a terminator (stop site) where it releases from the DNA 21 Translation of Proteins Series of three (triplets) bases in mRNA form codons each codon corresponds to a specific amino acid 23 Genetic Code 61 different codons (mRNA) code for 20 different amino acids (obviously some repeat) ONE codon AUG signals START and occurs at the beginning of every mRNA It codes for the amino acid methionine, so every proteins starts with this amino acid Three codons signals STOP and one of these will be found at the end of each mRNA UAA, UAG or UGA Notice: where different codons code for the same amino acid, the first two bases are often the same and the last differs. Because of this, the third base is often called the ‘wobble base’. It may help to protect against mutations in some cases Let’s Translate A Protein If our mRNA reads like this… AAAAGUAUGCGUUGGUGUGGUGGCGAUGCAGUAUGUUACUCAUAACCUAA Find the START codon (AUG) and break the sequence into codons (3 base sections) from that point… continue until you reach a STOP codon (UAA, UAG or UGA) Then read the codons to determine the appropriate amino acid to use next) AAAAGU AUG CGU UGG UGU GGU GGC GAU GCA GUA UGU UAC UCA UAA CCUAA Met- Arg- Trp- Cys- Gly- Gly- Asp- Ala- Val- Cys- Tyr- Ser- STOP tRNA Met This end holds the amino acid and is specific – it only holds ONE PARTICULAR amino acid type (MET in this case). When carrying it’s amino acid a tRNA is said to be charged A This end, called the anticodon, is complimentary to the codon on the mRNA. (following base pair rules) G 26 C A U G A G C A C G C Translation begins when the two subunits of a ribosome to mRNA and find a start codon (usually AUG, which codes methionine) first tRNA, carrying an amino, binds in the ribosome to the mRNA by the anticodon The next codon position if filled by the appropriate charged tRNA The peptide bond forms between the two amino acids After forming the peptide bond, the ribosome releases the first (now empty) tRNA The amino acid it once carried is now attached to the amino acid on the next tRNA PA The ribosome moves up the mRNA so that the remaining tRNA is in the first site and another codon is positioned in the second site A new charged tRNA binds in the open position. The growing polypeptide is added to the new amino acid by forming a peptide bond The process repeats so that one amino acid is added at a time to the growing polypeptide (which is always anchored to a tRNA bound within the ribosome) The polypeptide continues to grow until the ribosome reaches a stop codon At the stop codon, the polypeptide chain is released from the last tRNA and is complete The two subunits of the ribosome detach from each other and the mRNA Regulation of Gene Expression A gene may be constitutive: gene which is always turned on If not ALWAYS ON a gene must be regulated in some fashion… Promoter: region at beginning of gene which binds RNA Polymerase (sometimes thought of as a ‘switch’ zone) 31 Regulation of Gene Expression Repression: gene expression is lowered. mediated by repressor proteins which block binding of RNA polymerase at the operator site (on the DNA) near the promoter Induction: process of turning a gene on often an inducer molecule removes a repressor from the DNA 33 Regulating Groups of Genes operons: a group of genes located together in the DNA and which are regulated together. When an operon is activated all genes in the region tend to be made. 34 Mutations Mutation: a change in the base sequence of DNA May be considered "silent" if it doesn’t cause a change in amino acids (often in the third position of a codon) 37 Types of Mutations point mutation: single base substitution Substituting a single base pair is the most common form of mutation. This can result in several problems… (see next slides) 38 Types of Mutations if mutation changes the amino acid as in this example (Gly to Ser)… Then it is a missense mutation Nonsense Mutation If causes a stop codon Frameshift mutation: adding or deleting bases (usually cause a string of incorrect amino acids and/or a premature stop codon) Sources for Mutation can be spontaneous (natural errors of replication) can be induced by a mutagen (any agent that causes mutation) chemical radiation (UV or ionizing) 41 Identifying Mutagens Identifying Mutagens Ames Test: identifies potential human carcinogens by measuring mutagenesis in bacteria uses an auxotroph: a mutant strain of bacteria having a nutritional requirement not found in the parent bacterial strain Normal parent bacteria = prototroph, doesn’t have this nutritional restriction expose the auxotroph to the mutagen, if it regains prototrophy, then it has been mutated 43 Gene Transfer & Recombination Transformation: the uptake and incorporation of genes from naked DNA. first described & demonstrated by Frederick Griffith (1928 – England) DNA identified as ‘transforming principle’ by Avery, MacLeod, and McCarty (1944) Recombination: the insertion of new genes into a genome 46 Transformation: the uptake and incorporation of genes from naked DNA. in this case “dangerous” genes from dead bacteria taken in by previously benign bacteria Introducing DNA Into Cells Conjugation: (fig. 8.26, p. 237) transmission of genetic material via cell-cell contact. requires a conjugative plasmid (small circular DNA) using a sex pilus 49 Bacterial Conjugation as a method for inserting new DNA F-factor = fertility factor, genes with information to make a sex pilus Introducing DNA Into Cells Transduction: viral mediated transfer of DNA (fig. 8.28, p. 239). Two kinds: generalized (any gene) specialized (always a specific gene) 51 Plasmids and Transposons Plasmid: smaller, extrachromosomal, closed-circular DNA molecules which are self-replicating. do not contain essential genes can carry genes which give a special advantage to the cell examples: special catabolic pathways, antibiotic resistance Transposons: small segments of DNA that can move from one region of DNA to another ("jumping genes") (700 – 40,000bp) discovered in corn by Barbara McClintock (won Nobel Prize) simplest forms are called insertion sequences 53