Genetics Lecture 2 Final 2024 PDF

General Microbiology and Immunology Bacterial Genetics Lecture 2  Regulation of gene expression Lecture 2 Mutation outline Transfer of genetic elements Regulation of gene expression Because protein synthesis requires a huge amount of energy, cells...

General Microbiology and Immunology Bacterial Genetics Lecture 2  Regulation of gene expression Lecture 2 Mutation outline Transfer of genetic elements Regulation of gene expression Because protein synthesis requires a huge amount of energy, cells save energy by making only those proteins needed at a particular time. Constitutive (house-keeping) versus inducible genes. Structural versus regulatory genes. Lac operon as an example of gene regulation in prokaryotes Regulation of bacterial gene expression Because protein synthesis requires a huge amount of energy, cells save energy by making only those proteins needed at a particular time. Many genes, perhaps 60–80%, are not regulated but are instead constitutive (house-keeping), meaning that their products are constantly produced at a fixed rate. Usually these genes, which are effectively turned on all the time, code for enzymes that the cell needs in fairly large amounts for its major life processes. Glycolysis enzymes are examples. The production of other enzymes is regulated so that they are present only when needed. Regulation of gene expression is the ability of the cell to control or regulate protein synthesis from its DNA. Gene regulation mostly occurs at transcription level rather than translation, because transcription is the first step in biological information flow, so it is simple and efficient to control gene expression at this point. Genes are classified into structural genes (protein coding genes i.e. code for proteins) and regulatory genes (regulate the synthesis of proteins coded by the structural genes i.e. regulate the structural genes). Operon is a cluster of consecutive genes whose expression is under the control of a single operator. All of the genes in an operon are transcribed as a single unit yielding a single mRNA. The operator is located downstream of the promoter where synthesis of mRNA is initiated.  Regulation of gene expression Pre-transcriptional control Post-transcriptional control Negative control Positive control (activation) Enzyme repression Enzyme induction Lac operon Negative control of transcription: a control that prevents transcription through the action of a repressor. Positive control of transcription: in this case the regulatory protein is an activator that activates the binding of RNA polymerase to DNA.  Enzyme repression Enzyme induction Negative regulation Repressible operon Inducible operon Negative regulation of gene expression (cont.) The default position of a repressible gene is ON The default position of an inducible gene is OFF  Lac operon Lac operon is a group of genes that regulate the transport and metabolism of lactose in E. coli and many other enteric bacteria. Lac operon is a very basic model of gene regulation in E. coli and it has become a famous example of prokaryotic gene regulation. Gene regulation of lac operon was the first genetic regulatory mechanism to be understood clearly. Lac operon is an inducible operon. Lactose is converted in the cell to its isomer allolactose which is the inducer for these genes.  Lac operon (cont.) Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Three of the enzymes for lactose metabolism are grouped in the lac operon: lacZ, lacY, and lacA. lacZ encodes an enzyme called β-galactosidase, which digests lactose into its two constituent sugars: glucose and galactose. lacY codes for a permease that helps to transfer lactose into the cell. Finally, lacA codes for a trans-acetylase which metabolizes certain disaccharides other than lactose. Transcription of the lac operon normally occurs only when lactose is available for the cell to digest and glucose is absent. Presumably, this avoids wasting energy in the synthesis of enzymes for which no substrate is present. A single mRNA transcript includes all three enzyme- coding sequences and is called polycistronic (cistron=gene).  Positive regulation Regulation of the lactose operon also depends on the level of glucose in the medium, which in turn controls the intracellular level of the small molecule cyclic AMP (cAMP), a substance derived from ATP that serves as a cellular alarm signal. Enzymes that metabolize glucose are constitutive, and cells grow at their maximal rate with glucose as their carbon source because they can use it most efficiently. When glucose is no longer available, cAMP accumulates in the cell. The cAMP binds to the allosteric site of catabolic activator protein (CAP). CAP then binds to the lac promoter, which initiates transcription by making it easier for RNA polymerase to bind to the promoter. Thus, transcription of the lac operon requires both the presence of lactose and the absence of glucose.  Mutation Change in the nucleotide sequence of the DNA change in amino acid sequence of the resulting polypeptide change in the phenotype of the bacterial cell. Mutations can be either spontaneous or induced. Induced mutations are those that are due to agents in the environment and include mutations made deliberately by humans. They can result from exposure to natural radiation (e.g. cosmic rays) that alters the structure of bases in the DNA. In addition, a variety of chemicals, including oxygen radicals, can chemically modify DNA. For example, oxygen radicals can convert guanine into 8-hydroxyguanine, and this causes mutations. Spontaneous mutations are those that occur without external intervention. The bulk of spontaneous mutations result from occasional errors in the pairing of bases by DNA polymerase during DNA replication. A point mutation is when a single base pair in DNA sequence is altered. Point mutations are caused by base-pair substitutions in the DNA or by the insertion or deletion of a single base pair. The consequences of various base-pair substitutions Point mutations due to base-pair substitution can have one of three effects. First, the base substitution can be a silent mutation where the altered codon corresponds to the same amino acid. This is due to the degeneracy of the genetic code. Second, the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid at a specific site. This is called a missense mutation because the informational “sense” (precise sequence of amino acids) in the polypeptide has changed. If the change is at a critical location in the polypeptide chain, the protein could be inactive or have reduced activity. However, not all missense mutations necessarily lead to nonfunctional proteins.  The consequences of various base-pair substitutions (cont.) 1. Silent mutation 2. Missense mutation 3. Or third, the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal. In this case, the change is from a sense (coding) codon to a nonsense codon. Unless the nonsense mutation is very near the end of the gene, the product is considered truncated or incomplete. Truncated proteins are completely inactive or, at the very least, lack normal activity. Base-pair substitution (cont.) Other terms are common in microbial genetics to describe the type of base-pair substitution in a point mutation. Transitions are mutations in which one purine base (A or G) is A→G OR C→T substituted for another purine, or one pyrimidine base (C or T) is substituted for another pyrimidine. Transversions are point mutations in A/G → C/T which a purine base is substituted for a pyrimidine base, or vice versa. Mispairing (mismatching) occurs when A mismatches with C or a G A….C OR G….T mismatches with T.  Frameshift mutation Because the genetic code is read from one end of the nucleic acid in consecutive blocks of three bases (codons), any deletion or insertion of a single base pair results in a shift in the reading frame. These frameshift mutations often have serious consequences. Single base insertions or deletions change the primary sequence of the encoded polypeptide, typically in a major way. Such microinsertions or microdeletions can result from replication errors. Insertion or deletion of two base pairs also causes a frameshift; however, insertion or deletion of three base pairs adds or removes a whole codon. This results in addition or deletion of a single amino acid in the polypeptide sequence. Although this may well be deleterious to protein function, it is usually not as bad as a frameshift, which scrambles the entire polypeptide sequence after the mutation point.  Reversions/Back mutation Sometimes a second mutation can reverse the effect of an initial mutation. Furthermore, all organisms possess a variety of systems for DNA repair. G Mutation A Back Mutation G......... C T C Wild-type Revertant Mutant  Ames test using bacteria as screening agents for potential mutagenicity of chemicals The Ames test (named for the biochemist Bruce Ames who developed the test) makes practical use of detecting revertants in large populations of mutant bacteria to test the mutagenicity of potentially hazardous chemicals. Many known mutagens have been found to be carcinogens, substances that cause cancer in animals, including humans. A standard way to test chemicals for mutagenesis in the Ames test is to look for an increased rate of back mutations (reversion) in auxotrophic strains of bacteria. The Ames test assays for back mutations instead of forward mutations (generating auxotrophs from the wild type) because revertants can be more easily selected. Cells of such an auxotroph do not grow on a medium lacking the required nutrient (for example, an amino acid). Ames test (cont.) using bacteria as screening agents for potential mutagenicity of chemicals  Ames test (cont.) using bacteria as screening agents for potential mutagenicity of chemicals The Ames test is based on the observation that exposure of mutant bacteria to mutagenic substances may cause new mutations that reverse the effect (the change in phenotype) of the original mutation. These are called reversions. Specifically, the test measures the reversion of histidine auxotrophs of Salmonella (so-called his- cells, mutants that have lost the ability to synthesize histidine) to histidine-synthesizing cells (his+) after treatment with a mutagen. Bacteria are incubated in both the presence and absence of the substance being tested. Because animal enzymes must activate many chemicals into forms that are chemically reactive for mutagenic or carcinogenic activity to appear, the chemical to be tested and the mutant bacteria are incubated together with rat liver extract, a rich source of activation enzymes. If the substance being tested is mutagenic, it will cause the reversion of his- bacteria to his+ bacteria at a rate higher than the spontaneous reversion rate. The number of observed revertants indicates the degree to which a substance is mutagenic and therefore possibly carcinogenic.  Transfer of genetic elements Three mechanisms of genetic exchange are known in prokaryotes: (1) transformation, in which free DNA released from one cell is taken up by another; (2) transduction, in which DNA transfer is mediated by a virus; and (3) conjugation, in which DNA transfer requires cell-to-cell contact and a conjugative plasmid in the donor cell. The fate of transferred DNA. Whether it is transferred by transformation, transduction, or conjugation, DNA that enters the cell by horizontal gene transfer faces three possible fates: (1) It may be degraded by restriction enzymes; (2) it may replicate by itself (but only if it possesses its own origin of replication, such as a plasmid or phage genome); or (3) it may recombine with the host (recipient) chromosome. Vertical versus horizontal gene transfer Transformation Transformation (competence) is a genetic transfer process by which free DNA is incorporated into a recipient cell and brings about genetic change. Several prokaryotes are naturally transformable, including certain species of both Gram-negative and Gram- positive bacteria.  Transformation (cont.) Because the bacterial DNA is present in the cell as a large single molecule, when a cell is gently lysed, its DNA pours out. Then, this bacterial chromosome is easily broken down because of its extreme length and is converted into fragments having a typical transformable size (about 10 genes each). A recipient competent cell can then incorporate one or a few these DNA fragments. A cell that is able to take up DNA and be transformed is said to be competent, and this capacity is genetically determined. Competence in most naturally transformable bacteria is regulated, and special proteins play a role in the uptake and processing of DNA. These competence-specific proteins include a membrane- associated DNA-binding protein. Transformation (cont.) Examples of bacterial species which can undergo natural transformation include Bacillus spp. and Streprococcus spp. By contrast, many bacteria are poorly transformed, if at all, under natural conditions. For example, Escherichia coli and many other Gram-negative bacteria fall into this category. However, if cells of E. coli are treated with high concentrations of Ca2+ and then chilled, they become adequately competent (chemically competent cells are transformed by heat shock). Transformation (cont.) Electroporation is a physical technique that is used to get DNA into organisms that are difficult to transform, especially those with thick cell walls. In electroporation, cells are mixed with DNA and then exposed to brief high- voltage electrical pulses. This makes the cell envelope permeable and allows entry of the DNA.  Conjugation DNA transfer via physical cell contact. Donor cell with fertility factor (F+) Recipient cell (F-) First step is the establishment of direct cell-to-cell contact and the formation of a sex pilus (not sexual reproduction) or conjugation tube (extracellular filamentous structure generated by the donor cell). The second step is the delivery of extrachromosomal DNA (plasmid) to the recipient cell after its replication in the donor cell. Transduction In transduction, bacterial DNA is transferred from a donor cell to a recipient cell inside a virus that infects bacteria, called a bacteriophage, or phage. Thank you