BIO425 Chapter 6. Genetics of Bacteria-1 PDF

Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 1 MICROBIOLOGY LECTURE for the immune system Main Topic Sources: PPT, Lecture Videos, Book to...

Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 1 MICROBIOLOGY LECTURE for the immune system Main Topic Sources: PPT, Lecture Videos, Book to detect the bacteria). DNA IS THE GENETIC MATERIAL Polysaccharide capsule Genetic material adds to the virulence of ○ Hereditary substance in the cell the microorganism. ○ Carries all information specific to an organism Mouse died. ○ DNA (Deoxyribonucleic acid) ○ RBA (Ribonucleic acid) Figure C: Bacteria from Figure D: Heat-killed S the S strain were killed strain was added to the Major 20th Century Events by heat. R strain bacteria. 1928: Frederick Griffith showed that bacteria Heat-killed strain cannot The blend of two strains can get DNA through a process called cause infection. harmed the mouse. transformation. Mouse is unharmed. Live S and R strains of the pneumococcal bacteria were isolated from the blood of the dead mouse. ○ Griffith concluded that the type R had been transformed into the little S strain by the transforming principle. Bacterial transformation: the process by which a bacterium can get and use new genetic ○ Griffith made use of pneumococcal material coming from its bacteria: Streptococcus pneumoniae surroundings. Causative agent of pneumonia, During bacterial otitis media, and sepsis (blood transformation, a non infection) disease-causing bacterium Can cause diseases only if they (avirulent strain) can turn into are encapsulated; Without the a disease-causing bacteria capsule, it cannot cause (virulent strain). infection. Transformation can occur even ○ Smooth (S strain) - virulent strain if the disease-causing strain is Virulent - can cause harmful dead, implying dead bacterial diseases to the host transformation happens when ○ Rough (R strain) - avirulent strain the non disease-causing Avirulent - cannot cause bacteria inherits genetic disease material from the disease-causing bacteria. Figure A: Strain covers Figure B: Strain does not itself with the have the protective 1944: Oswald Avery, Colin Macleod, and Maclyn polysaccharide capsule. shield (capsule). Mccarty showed that DNA can transform bacteria, demonstrating that DNA is the The capsule makes the Immune system is now hereditary material. bacteria virulent to able to detect the protect it presence of the bacteria. From the host’s immune Mouse is unharmed. system (will be difficult Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 2 ○ They made various DNA radioactive with 32𝑃 or they labeled its protein with 35𝑆. ○ They mixed radioactive virions with Escherichia coli and incubated the mixture for a few minutes. Virions attach to the E. coli and initiate the infection process. ○ Several centrifugations were done. ○ This experiment settled the long ○ Built on the foundation of Griffith’s standing debate about the study which they identified as the composition of genes thereby allowing transforming principle that allowed scientists to investigate the molecular bacteria to acquire characteristics mechanisms by which genes function from one another. in organisms. ○ Conducted to know what makes ○ Hershey and Chase were able to transformation possible. confirm that DNA is the genetic ○ First used the process of elimination material. to determine that neither carbohydrates nor lipids were responsible. PROKARYOTIC GENOME ○ Then, tested proteins, RNA, and DNA. Bacterial genomes are studied to better ○ Scientists knew that the bacterial understand their metabolic capabilities. transformation should take place We also get to know their ability to cause unless they disable the transforming diseases and their capacity to survive in principle. extreme environments. ○ Enzymes were added such as proteases, RNases, and DNases in Structure of Bacterial Genome order to destroy various components of the bacterial mixture. DNases RNases Proteases Directed to DNA Directed to Directed to which is the RNA. proteins. transforming principle. Bacterial genome consists of macromolecules ○ Some steps were transformed because and genes. the transforming principle, DNA, was DNA is double-stranded which forms a helix. not destroyed. DNA is a double helical structure. Each deoxynucleotide contains a phosphate, 1952: Hershey-Chase Experiment, also called 5-carbon sugar (deoxyribose), 4 nitrogenous the Waring Blender Experiment, by Alfred bases (adenine, guanine, cytosine, thymine). Hershey and Martha Chase provided concrete ○ Phosphate and the sugar make up the evidence that genes were made of DNA. backbone of each DNA strand. ○ Bases are responsible for holding the two strands together which is made possible by hydrogen bonds existing between the nitrogenous bases. ○ Order of the bases in the DNA strand contains the coded genetic information. Genetics Elements: ○ Chromosomes Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 3 ○ Plasmids RNA polymerase, an enzyme DNA of most bacteria is contained in a single that synthesizes RNA from the circular molecule called the bacterial coding region of the gene. chromosome. Three informational macromolecules in cell: ○ The chromosome, along with several DNA, RNA, Protein proteins and RNA molecules, forms an irregularly shaped structure called the nucleoid. This sits in the cytoplasm of the bacterial cell. Bacteria often contain plasmids which are small circular DNA molecules. ○ Bacteria can easily pick up new ○ Central dogma - key instructions in plasmids from other bacterial cells DNA are converted into the functional which is made possible through the product (protein). process of conjugation or even from Explains the flow of genetic the environment. information from DNA to RNA ○ They can also readily lose plasmids. and then to protein. E.g. Bacterium divides in two ○ First proposed by Francis Crick, who is and one of the daughter cells also the discoverer of the structure of might miss out on getting a DNA. plasmid. DNA RNA Macromolecules and Genes Functional unit of genetic information is the Contains the information Serves as the messenger gene. needed to make all of our that carries the Genes are in cells and are composed of DNA. proteins. information to the A nucleic acid strand is inherently directional. ribosomes. Ribosomes for protein synthesis. Ribosomes serve as factories in the cell ○ 5’ (five prime) to 3’ (three prime) where the information is ○ 3’ (three prime) to 5’ (five prime) translated from a code ○ 5’ end has a free hydroxyl or phosphate into the functional group on a 5’ carbon. product. ○ 3’ end has a free hydroxyl or phosphate group on a 3’ carbon. ○ The process by which DNA instructions ○ A bacterial gene has up to 90% coding are converted into RNA to protein is sequence. referred to as gene expression. Continuous coding sequences ○ Gene expression has two key stages: are called exons. Transcription and Translation Intervening non-coding DNA is transcribed into RNA sequences are called introns. and RNA is translated into ○ Promoter is a regulatory region of DNA proteins. located upstream towards the 5’ region Transcription - the information of a gene providing a control point for in the DNA of every cell is regulated gene transcription. converted into small portable Promoter contains specific DNA RNA messages. sequences that are recognized Translation - the RNA by proteins known as messages travel from where transcription factors. the DNA is in the cell nucleus to These factors bind to the the ribosomes where they are promoter sequences of protein read to make specific proteins. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 4 Complementary Base Pairing: Adenine and Thymine; Cytosine Summary: Three stages of genetic Information flow and Guanine Replication Transcription Translation Complementary base pairs result in the production of two DNA is Information Information in complementary strands of DNA. duplicated from DNA is RNA is used to ○ Transcription is a process by which the transferred into build information in a strand of DNA is RNA polypeptides. copied into a new molecule of mRNA messenger RNA (mRNA). (messenger Synthesis of RNA under the RNA) encodes direction of DNA polypeptides Carried out by an enzyme referred to as the RNA tRNA (transfer polymerase. RNA) plays role in protein There are also a number of synthesis accessory proteins called transcription factors. rRNA ○ Prokaryotic transcription is a process (ribosomal in which mRNA transcripts of genetic RNA) material in prokaryotes are produced to plays role in be translated for the production of protein synthesis proteins. Occurs in the cytoplasm alongside with the process of translation. ○ Transcription and translation in the Synthesis of the three informational prokaryotes can occur simultaneously. macromolecules This is not possible with eukaryotes as they have a membrane-bounded nucleus in which transcription takes place. Translation, on the other hand, occurs in the cytoplasm. In prokaryotes, genetic material is not enclosed in a membrane-bounded nucleus thus, having access to ribosomes in the cytoplasm. ○ Replication is the process by which the double-stranded molecule is copied to produce two identical DNA molecules. During this process, the helix unwinds. Each strand of DNA serves as a template for replication. Most bacteria employ polycistronic The DNA polymerase organizes transcription. the assembly of the new DNA ○ Several genes of related function are strands so the nucleotides that transcribed into a single RNA molecule. make up the new strands are Eukaryotes: each gene is transcribed paired with partnered individually. nucleotides in the template strand. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 5 Prokaryotes: multiple genes may be transcribed together. THE DOUBLE HELIX Phosphodiester bond is the backbone of the nucleic acid present in life existing on Earth. ○ It is a covalent bond between phosphate and two sugars (hydroxyl groups). ○ Covalent bonds are said to be stronger than hydrogen bonds. Four nucleotides are found in DNA: ○ Adenine (A) Overall arrangement of Double Helix ○ Guanine (G) ○ Cytosine (C) ○ Thymine (T) Each deoxynucleotide contains a phosphate. Backbone of the DNA chain is alternating phosphates and the pentose sugar deoxyribose. Phosphates connect 3’ carbon of one sugar to 5’ carbon of the adjacent sugar. All cells and some viruses have DNA in double-stranded molecules. Two strands are antiparallel ○ Antiparallel - they have the same Major groove Minor groove chemical structure but are opposite in direction. Occurs where the Occurs where the ○ They are antiparallel because of the backbones are far apart. backbones are close hydrogen bonds. together. Two strands have complementary base sequences ○ The grooves twist around the molecule ○ Adenine always pairs with Thymine on opposite sides. Only two hydrogen bonds are They allow proteins to bind to present between these two. and recognize DNA sequences ○ Guanine always pairs with Cytosine from the outside of the helix. Three hydrogen bonds are Grooves expose the edges of present between these two. each base pair located inside ○ Held together by hydrogen bonds the helix which allows proteins Two strands form a double helix to chemically recognize specific DNA sequences. ○ Sugar-phosphate backbone Far apart in the major groove and close together in the minor groove. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 6 Supercoiled DNA: DNA is further twisted to They vary in length and in the save space number of DNA or RNA ○ Negative supercoiling: double helix is molecules underwound Linear and single-strand DNA: Decreases the number of base e.g. parvovirus pairs per turn. Double-stranded: e.g. ○ Positive supercoiling: double helix is adenovirus overwound Circular, single-stranded DNA: Increases the number of base e.g. plasmids, used for many pairs per turn. recombinant DNA processes ○ Expression of the strain on the strand. Circular, double-stranded DNA: ○ On the average, the helical repeat is e.g. baculovirus (insect about 10.5 base pairs per helical turn pathogenic viruses used for of DNA. pest controls) ○ Bacterial chromosomes are negatively ○ Plasmids are distinct from supercoiled. chromosomal DNA. ○ Supercoiling helps compact DNA in It can divide independently of order to fit into the cell. the bacterial chromosome. ○ This also loosens up DNA making it Naturally exist in bacterial cells easier to separate the two strands from and also occur in some each other which is considered eukaryotes. important in DNA replication. Genes carried in plasmids Relaxed DNA: DNA has number of turns provide bacteria with genetic predicted by number of base pairs advantages such as being Negative supercoiling is predominantly found antibiotic resistant. in nature Great majority are DNA gyrase: reduces supercoiling double-stranded; Most are ○ An essential bacterial enzyme that circular catalyzes the ATP-dependent negative Not extracellular, unlike viruses supercoiling of double-stranded closed ○ Organellar genomes or DNA are circular DNA. contained in organelles (e.g. ○ Gyrase belongs to a class of enzymes mitochondria, chloroplasts) and called topoisomerase. outside of the nucleus in eukaryotic ○ Involved in the control of topological cells transitions of DNA. ○ Transposable elements, also known as transposons or jumping genes, is the DNA sequence that can change its GENETIC ELEMENTS position within a genome, sometimes Chromosomes and Plasmids creating or reversing mutations and Genome: entire complement of genes in cell or altering cells' genetic identity with its virus genome size. ○ Complete set of genetic information in an organism ○ Provides all the information that the organism requires in order to function. Chromosome: main genetic element ○ Where genome is stored Other genetic elements include virus genomes, plasmids, organellar genomes, and transposable elements. ○ Viral genomes are diverse as they can RNA can be linear or circular be an RNA or DNA. ○ Linear, single stranded RNA molecule: Can be single or e.g. new tobacco mosaic virus double-stranded ○ Nonenveloped double-stranded RNA Can be linear or circular virus: e.g. reovirus (associated with respiratory enteric orphan virus) Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 7 ○ Circular, single-stranded RNA molecule: transposons bounded by hepatitis D (delta hepatitis; causes insertion sequence which liver infection) supply transposase, activity Hepatitis D only occurs on needed for transcription. people with hepatitis B Unit transposons - contain one Plasmids: genetic elements that replicate or more gene encoding independently of the host chromosome enzymes needed for ○ Small circular or linear DNA molecules transposition. ○ Range in size from 1 kbp to >1 Mbp Imperative conjugated typically less than 5% of the elements also in size of the chromosome addition to transposon ○ Carry a variety of nonessential, but functions. often very helpful, genes Contain genes for ○ Abundance (copy number) is variable conjugative transfer to ○ Have a wide range of lengths, from a new host cell and roughly one thousand DNA base pairs other genes (e.g. to hundreds of thousands of base antibiotic resistance pairs. genes) ○ These are important for bacterial evolution and adaptation to changing environments. FLOW OF GENETIC INFORMATION ○ Different plasmids can coexist in one Replication, Transcription, & Translation in bacterial cell. Prokaryotes Transposable elements PART 1. DNA Replication Two strands of double helix uncoil and are separated. ○ Each serves as a template for the synthesis of complementary strands according to the base pairing rules. The replication of chromosomal DNA begins at a single point, called the origin of replication. Synthesis of DNA occurs in the replication fork. ○ Place at which the DNA helix is ○ Segments of DNA that can move from unwound and the individual strands one site to another site on the same or are replicated. a different DNA molecule. Bidirectional Replication and the Replisome ○ Mobile DNA sequences that are widely distributed in prokaryotic and ○ DNA replication occurs in both eukaryotic genomes where they directions. represent a major course in genome ○ From the origin of replication in the evolution. circular DNA found in most bacteria. ○ Inserted into other DNA molecules. All the proteins involved in DNA replication ○ Transposable elements are rarely aggregate at the replication fork to form a documented in viruses. replication complex called the replisome. ○ Their contribution to the viral genome ○ Replisome is a large protein complex revolution remains largely unemployed. that carries out DNA replication starting at the replication stage. ○ Three main types: Insertion sequences, Transposons, Special ○ Contains enzymatic activities such as viruses helicase, primase, and DNA polymerase which creates a replication Insertion sequences - small fork to duplicate both the leading and genetic elements consisting of lagging strand. transposase genes racketed by inverted species. A Lagging DNA strand loops out from the leading strand and this enables the replisome Composite transposons - contains genes unrelated to Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 8 to move along both strands, pulling DNA through as replication occurs. It is the actual DNA, not the polymerase, that moves during the bacterial DNA replication. DNA Replication in Prokaryotes ○ Enzymes with suffix -ase ○ Helicase is responsible for unzipping the DNA. ○ DNA replication is semiconservative. Each of the two progeny double helices have one parental and one new strand. ○ Replication always proceeds from the 5’ end to the 3’ end. Nucleotides can only be added to the 3’ end of a growing chain. The Origin of Replication ○ Enzyme - DNA polymerase If the helicase unzips the DNA, the DNA polymerase adds nucleotides on RNA primer and the growing strand. ○ DNA ligase attaches the okazaki fragments of the lagging strand. ○ Okazaki fragments are small sections of DNA that are formed during downstream synthesis. Considered important as they allow both daughter strands to be synthesized which is necessary for their division. DNA Gyrase - Topoisomerase ○ It produces supercoiling. It relaxes tension which builds Leading Strand Lagging Strand up during DNA unwinding. Preventing DNA breakage Adds the nucleotides Adds groups of Proofreading in DNA continuously nucleotides, okazaki fragments, ○ DNA replication is extremely accurate discontinuously from 5’ Proofreading helps to ensure to 3’ direction. high fidelity. −8 −11 ○ Mutation rates in cells are 10 to 10 errors per base inserted. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 9 ○ Polymerase can detect mismatch ○ Recognized by sigma factor of RNA through incorrect hydrogen bonding. polymerase The mismatched base pairs ○ Transcription stops at specific sites Polymerase uses the 3’ end to called transcription terminators. 5’ ends of nucleus activity to Unlike DNA replication, transcription involves remove the incorrect pairing smaller units of DNA, from the 3’ end and the new ○ Often as small as a single gene strand. ○ Allows cell to transcribe different genes ○ Proofreading occurs in prokaryotes, at different rates. eukaryotes, and viral DNA replication Two regions within promoters that are highly systems. conserved: PART 2. RNA Synthesis: Transcription First part of gene expression Carried out by RNA polymerase (uses DNA as template) DNA to RNA ○ DNA synthesis requires dATP, dGTP, dTTP, and dCTP as substrates. ○ RNA requires ATP, GTP, CTP, and UTP. Natural nucleotide triphosphates (contains ribose) ○ Adenosine triphosphate (ATP) ○ Conserved regions - present across all ○ Guanosine triphosphate (GTP) organisms ○ Cytidine triphosphate (CTP) ○ Pribnow box: located 10 bases before ○ Thymidine triphosphate (TTP) the start of transcription (-10 region) ○ Uridine triphosphate (UTP) ○ -35 region: located -35 bases upstream Chain growth is 5’ to 3’ just as in DNA of transcription replication. Transcription: Termination ○ Termination of RNA synthesis is governed by a specific DNA sequence. Intrinsic terminators: transcription is terminated without any additional factors. Rho-dependent termination: Rho protein recognizes specific Only one of the two strands of DNA is DNA sequences and causes a transcribed by RNA polymerase for any gene. pause in the RNA polymerase. Genes are present on both strands of DNA. Prokaryotes often have genes clustered RNA polymerase has five different subunits. together. RNA polymerase recognizes DNA sites called ○ These genes are transcribed all at once promoters. as a single mRNA. ○ Promoter: site of initiation of An mRNA encoding group of co-transcribed transcription genes is called a polycistronic mRNA. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 10 ○ Polycistronic mRNA carries several ○ The initial RNA that is transcribed from open reading frames (ORF). the gene’s DNA template must be ○ Each of the ORF is translated into processed before it becomes immature proteins. messenger RNA (mRNA) that can direct the synthesis of proteins. Eukaryotic RNA Processing ○ Operon: a group of related groups co-transcribed on a polycistronic mRNA. Allows for expression of multiple genes to be coordinated. Transcription in Archaea and Eukarya RNA splicing RNA capping Poly(A) tail ○ The Archaea, like Bacteria, utilizes only a Takes place in Addition of Addition of single type of RNA polymerase to nucleus methylated 100-200 transcribe all genes. guanine to 5’ end adenylate Resembles eukaryotic Removes introns of mRNA residues polymerase II from RNA ○ Archaea have a simplified version of transcripts Cap protects the Stabilizes mRNA 5’ end of the and is required eukaryotic transcription apparatus. Performed by the primary RNA for translation Promoters and RNA polymerase spliceosome, transcript from are similar to eukaryotes. huge multi the attack by Prevents the Regulation of transcription has megadalton ribonucleases. degradation of major similarities with Bacteria. ribonucleoprotei RNA molecules ○ Eukaryotic genes have coding and n (RNP) which Cap is then noncoding regions. are found in recognized by Allows the Exons - coding sequences eukaryotic nuclei. eukaryotic mature initiation factors messenger RNA Introns - non-coding or Final mRNA involved in molecule to be intervening sequences consists of the assembling the exported from the Introns are rare in Archaea. remaining ribosomes on the nucleus and Introns are found in the tRNA sequences, mature mRNA translated into a and rRNA genes of Archaea. exons, which are prior to initiating protein by ○ Archaeal introns excised by special connected to one translation. ribosomes in the endonuclease. another through cytoplasm. the splicing process. Eukaryotic RNA processing: many RNA molecules are altered before they carry out their role in the cell  Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 11 Transcription in Prokaryotes process in which messenger RNA transcripts of genetic material in prokaryotes are produced, to be translated for the production of proteins Transcription involves three separate processes: initiation, elongation, and termination Transcription involves three separate processes: initiation, elongation, and termination Protein Synthesis: Translation Translation involves decoding mRNA and covalently linking amino acids together to form a polypeptide; this occurs at the ribosome. Polypeptides, Amino Acids, and the Peptide Bond Proteins play a major role in cell function ○ Catalytic proteins (enzymes) ○ Structural proteins Proteins are polymers of amino acids Amino acids are linked by peptide bonds to form a polypeptide Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 12 Translation & the Genetic Code Translation: the synthesis of proteins from RNA Genetic code: a triplet of nucleic acid bases (codon) encodes a single amino acid ○ Specific codons for starting and stopping translation ○ Degenerate code: multiple codons encode a single amino acid ○ Anticodon on tRNA recognizes codon Transfer RNA Transfer RNA: at least one tRNA per amino acid ○ Bacterial cells have 60 different tRNAs ○ Mammalian cells have 100–110 different tRNAs Specific for both a codon and its cognate amino acid tRNA and amino acid brought together by aminoacyl-tRNA synthetases ○ ATP is required to attach amino acid to tRNA tRNA is cloverleaf in shape Fidelity of recognition process between tRNA and aminoacyl-tRNA synthetase is critical Incorrect amino acid could result in a faulty or Wobble: irregular base pairing allowed at third non functioning protein position of tRNA Protein Synthesis Translation & the Genetic Code Ribosomes: sites of protein synthesis Stop codons: terminate translation (UAA, UAG, Thousands of ribosomes per cell Composed of and UGA) two subunits (30S and 50S in prokaryotes) Start codon: translation begins with AUG ○ S = Svedberg units Reading frame: triplet code requires translation ○ Combination of rRNA and protein to begin at the correct nucleotide E. coli has 52 distinct ribosomal proteins Shine–Dalgarno sequence: ensures proper reading frame Open reading frame (ORF): AUG followed by a number of codons and a stop codon in the same reading frame Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 13 ○ Quaternary structure : Number and 1. Initiation: two ribosomal subunits assemble with types of polypeptides that make a mRNA protein Begins at an AUG start codon 2. Elongation: amino acids are brought to the ribosome and are added to the growing polypeptide Occurs in the A and P sites of ribosome Translocation: movement of the tRNA holding the polypeptide from the A to the P site 3. Termination: occurs when ribosome reaches a stop codon Release factors (RF): recognize stop codon and cleave polypeptide from tRNA Ribosome subunits then dissociate Subunits free to form new initiation complex PROTEIN FOLDING & SECRETION and repeat process Denaturation: ○ Occurs when proteins are exposed to extremes of heat, pH, or certain chemicals ○ Causes the polypeptide chain to unfold ○ Destroys the secondary, tertiary, and/or quaternary structure of the protein The biological properties of a protein are usually lost when it is denatured Most polypeptides fold spontaneously into their active form ○ Some require assistance from molecular chaperones or chaperonins for folding to occur ○ They only assist in the folding; they are not incorporated into protein ○ Can also aid in refolding partially denatured proteins Polysomes: a complex formed by ribosomes simultaneously translating mRNA Protein structures Once formed, a polypeptide folds to form a more stable structure. ○ Secondary structure : Interactions of the R groups force the molecule to twist and fold in a certain way ○ Tertiary structure : Three-dimensional shape of polypeptide Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 14 Signal sequences: found on proteins requiring transport from cell ○ 15–20 residues long ○ Found at the beginning of the protein molecule ○ Signal the cell's secretory system (Sec system) ○ Prevent protein from completely folding Secretion of folded proteins: the Tat system Proteins that fold in the cytoplasm are exported by a transport system distinct from Sec, called the Tat protein export system ○ Iron–sulfur proteins ○ Redox proteins Inducible Genes β-Galactosidase Enzyme Secretion of proteins: types I through VI Inducible enzyme functions in a catabolic ○ All are a large complex of proteins that pathway form channels through membranes Inducible enzymes are present only when their ○ Types II and V depend on Sec or Tat substrate (inducer - effector molecule) is ○ Types I, III, IV, and VI do not require Sec available or Tat β-galactosidase reaction catalyzed is lactose hydrolysis into galactose and glucose Two Approaches to Regulation Regulation of gene expression Repressible Genes ○ transcription initiation Enzymes that function in biosynthetic ○ transcription elongation pathways are products of repressible genes ○ Translation Generally these enzymes are always present Alter activity of enzymes and proteins unless the end product in the biosynthetic ○ Posttranslational pathway is available Three domains of life differ in genome structure and regulatory mechanisms used Control of Transcription Initiation by Regulatory Proteins Induction and repression occur because of the activity of regulatory proteins and DNA binding domains These proteins either inhibit transcription (negative control) or promote transcription (positive control) Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 15 TRANSCRIPTIONAL CONTROL Presence of allolactose binds repressor – no NEGATIVE TRANSCRIPTIONAL CONTROL longer binds operator ○ Binding of regulatory protein at DNA regulatory site inhibits initiation of transcription ○ mRNA expression is reduced ○ Repressor proteins ○ exist in active and inactive forms ○ inducers/corepressors alter activity of repressor by binding POSITIVE TRANSCRIPTIONAL CONTROL ○ Binding of a regulatory protein at a regulatory region on DNA promotes transcription initiation Lac operon – a negatively controlled inducible operon ○ mRNA synthesis is increased ○ Activation ○ inactive protein is activated by inducer ○ active protein is inactivated by inhibitor Examples of Transcriptional Control Negative Control of Lactose (Lac) Operon Inducible genes ○ three structural genes coding for lactose uptake and metabolism ○ lac repressor (lacI) binds operator inhibits transcription Enzymes normally not produced unless lactose present lac Repressor Tetramers of repressor form and bind to three operator sites (O1, O2, O3) Bends DNA, prevents RNA polymerase from accessing promoter Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 16 Trp Operon – a negatively controlled repressible operon Mutations: Their Chemical Basis and Effects Stable, heritable changes in sequence of bases in DNA ○ point mutations are the most common from alteration of single pairs of nucleotide from the addition or deletion of nucleotide pairs larger mutations are less common insertions, deletions, inversions, duplication, and translocations of nucleotide sequences Spontaneous Mutations Arise without exposure to external agents May result from errors in DNA replication ○ due to base tautomerization resulting in transition and transversion mutations ○ due to insertion or deletion of nucleotides May also result from the action of mobile genetic elements such as transposons Mutations Heritable change in DNA sequence that lead to a change in phenotype ○ observable traits Mutant ○ Strain of any cell or virus differing from parental strain in genotype nucleotide sequence of genome Wild-Type Strain ○ Strain isolated from nature Induced Mutations Made environmentally or deliberately Can result from exposure to natural radiation or oxygen radicals Caused by agents that directly damage DNA ○ base analogs Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 17 structurally similar to normal Missense Mutation bases Amino acid changed mistakes occur when they are Polypeptide altered incorporated into growing Nonsense Mutation polynucleotide chain Codon becomes stop codon ○ DNA modifying agents Polypeptide incomplete alter a base causing it to mispair ○ intercalating agents distort DNA to induce single nucleotide pair insertions and deletions Frameshift Mutations Deletions or insertitions that result in shift in the reading frame ○ Result in complete loss of gene Effects of Mutations function Wild type Deletions or insertions of fewer than 3 base ○ most prevalent form of gene pairs cause shift in reading frame Forward mutation Very deleterious and yield mutant phenotypes ○ wild type → mutant form resulting from the synthesis of nonfunc- tional Reversion mutation proteins ○ mutant phenotype → wild type produce a stop codon so that the peptide phenotype product is shorter as well as different in suppressor mutation sequence occurs when the second mutation is at a different site than the original mutation Mutations in Protein Genes Point mutations ○ Change only one base pair ○ Lead to a single amino change in a protein, incomplete protein, or no change at all Silent Mutation Does not affect the amino acid sequence Other types of Mutations Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 18 polymerase I, and DNA Conditional Mutations ligase joins the Expressed only under certain environmental fragments conditions ○ can remove thymine ○ E.g. conditional lethal mutation of dimers and repair bacteria culture @ high temp almost any other injury the mutant would grow that produces a normally at cooler detectable distortion in temperatures but would die at DNA. high temperatures. Auxotrophic Mutant Unable to make essential macromolecule e.g. amino acid, nucleotide ○ Has conditional phenotype unable to grow on medium lacking that molecule but grows when the molecule is provided ○ Wild-type strain from which it arose is called a prototroph DNA Damage & Repair DNA Repair Mechanisms 1. Proofreading - correction of errors in base pairing made during replication - errors corrected by DNA polymerase base excision repair 2. Mismatch Repair ○ employs enzymes - mismatch correction enzyme scans called DNA glyco- newly synthesized DNA for mismatched sylases. pairs remove - mismatched pairs removed and damaged replaced by DNA polymerase and DNA bases yielding ligase apurinic or - depends on the ability of mismatch apyrimidinic repair enzymes to distinguish between (AP) sites. parental and newly synthesized DNA ○ AP endonuclease strands. recognize the 3. Excision Repair damaged DNA - Corrects damage that causes and nick the distortions in double helix back- bone at - Two types of repair systems: the AP site both remove the damaged ○ DNA polymerase I portion of the DNA strand and removes the damaged use the intact complementary region, using its 5’ to 3’ strand as a template to exonuclease activity. synthesize new DNA It then fills in nucleotide excision repair the gap, and ○ UvrABC endonuclease DNA ligase removes damaged joins the DNA nucleotides and a few fragments. nucleotides on either side of the lesion. ○ The resulting single-stranded gap is filled by DNA Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 19 4. Direct Repair DNA Methylation - Corrects thymine dimers and alkylated bases Used by E. coli mismatch repair system to - Photoreactivation distinguish old DNA strands from new DNA used to directly repair thymine strands dimers ○ old DNA (template strand) methylated; thymines separated by new DNA not methylated photochemical reaction ○ the repair system cuts out the catalyzed by photolyase mismatch from the unmethylated requires visible light and is strand catalyzed by the enzyme catalyzed by DNA methyltransferases photolyase. - Direct repair of alkylated bases catalyzed by alkyltransferase or methylguanine methyltransferase damage to guanine from mutagens such as methyl-nitrosoguanidine can be repaired directly. 5. Recombinational Repair - Repairs DNA with damage in both strands - recombination with an undamaged molecule in rapidly dividing cells, another copy of chromosome is SOS Response often available Inducible repair system (a global control - RecA protein cuts a piece of template DNA network) from a sister molecule and puts it into the gap Used to repair excessive damage that halts or uses it to replace a damaged strand. replication, leaving many gaps ○ repressor LexA destroyed, neg. regulated genes activated over 50 genes are activated when a transcriptional repressor protein called LexA is destroyed. ○ Once LexA is destroyed, these genes are transcribed and the SOS response ensues. RecA protein ○ Initiates SOS response and recombination repair Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 20 ○acts as protease, destroying LexA ○ can integrate reversibly into the host repressor protein, increasing chromosome (episomes) production of excision repair enzymes Conjugative plasmids (F plasmid) can transfer DNA polymerases IV and V synthesize copies of themselves to other bacteria during unrepaired DNA conjugation lack proofreading activity. highly error prone and results in the generation of numerous mutations. Creating Additional Genetic Variability Mutations are subject to selective pressure ○ each mutant form that survives becomes an allele, an alternate form of a gene Recombination is the process in which one or more nucleic acids are rearranged or combined to produce a new nucleotide sequence (recombinants) F factors contain the information for formation Horizontal Gene Transfer in Bacteria and Archaea of sex pilus HGT differs from vertical gene transfer ○ attach F+ cell to F- cell for DNA transfer ○ transfer of genes from one during bacterial conjugation independent, mature organism to F factors have insertion sequences (IS) another ○ assists in plasmid integration stable recombinant has F+ x F- Mating characteristics of donor and A copy of the F factor is transferred to the recipient recipient and does not integrate into the host Important in evolution of many species chromosome ○ expansion of ecological niche, Donor genes usually not transferred increased virulence F factor codes for sex pilus ○ occurs in the three mechanisms ○ Type IV secretion system that makes evolved by bacteria to create contact between cells that DNA moves recombinants across ○ genes can be transferred to the same or Plasmid is replicated by rolling circle method different species 1. Bacterial Conjugation J. Lederberg and E. Tatum demonstrated the transfer of genes between bacteria that depends on ○ direct cell to cell contact mediated by the F pilus ○ unidirectional DNA transfer from donor to recipient Bacterial Plasmids HFr Conjugation Donor HFr cell has F factor integrated into its chromosome Small, autonomously replicating DNA Donor genes are transferred to recipient cell molecules A complete copy of the F factor is usually not ○ can exist independently from host transferred chromosome Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 21 Gene transfer can be clockwise or DNA Uptake in Bacterial Transformation counterclockwise Protein system allows DNA to move across cell walls Gram-negative ○ PilQ aids in movement across outer membrane ○ Pilin complex (PilE) moves DNA across periplasm and peptidoglycan ○ ComE is DNA binding protein ○ N is nuclease that degrades one strand ○ ComA forms transmembrane channel Similar system in Gram-positive. 3. Transduction The transfer of bacterial genes by viruses Viruses (bacteriophages) can carry out the lytic cycle (host cell is destroyed) or viral DNA F’ Conjugation integrates into the host genome (becoming a Result when the F factor incorrectly leaves the latent prophage) host chromosome Some of the F factor is left behind in the host chromosome Some host genes have been removed along with some of the F factor these genes can be transferred to a second host cell by conjugation 2. Bacterial Transformation F. Griffith demonstrated transformation Generalized Transduction Uptake of naked DNA by a competent cell Any part of bacterial genome can be transferred followed by incorporation of the DNA into the ○ Occurs during lytic cycle of virulent recipient cellÕs genome phage During viral assembly, fragments of host DNA mistakenly packaged into phage head ○ generalized transducing particle Carried out only by temperate phages that have established lysogeny Only specific portion of bacterial genome is transferred Occurs when prophage is incorrectly excised Drug Resistance An increasing problem ○ once resistance originates in a population it can be transmitted to other bacteria Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 22 ○ a particular type of resistance ○ can be transferred to other cells by mechanism is not confirmed to a conjugation, transduction, and single class of drugs transformation Microbes in abscesses or biofilms may be ○ can carry multiple resistance genes growing slowly and not be susceptible Resistance mutants arise spontaneously and are then selected Drug Resistant “Superbug” A methicillin-resistant Staphylococcus aureus (MRSA) that developed resistance to vancomycin ○ this new vancomycin-resistant S. aureus (VRSA) was also resistant to most other antibiotics ○ isolated from foot ulcers on a diabetic patient ○ Acquired from conjugation with Composite transposons vancomycin-resistant enterococci ○ contain genes for antibiotic resistance (VRE) were isolated from same patient - some have multiple resistance genes These drug resistant organisms are a serious can move rapidly between threat to human health plasmids and through a bacterial population Gene cassettes ○ sets of resistance genes ○ can exist as separate genetic elements ○ can be part of transposon, integron, or chromosome POST CREDIT FUN Congrats for reaching the end of this trans!! Good luck again makakapasa tayo claim it The Origin and Transmission of Drug Resistance Immunity genes ○ resistance genes that exist in nature to protect antibiotic producing microbes from their own antibiotics Horizontal gene transfer ○ transferred immunity genes from antibiotic producers to non-producing microbes Resistance genes can be found on: ○ bacterial chromosomes ○ plasmids ○ transposons ○ integrons When found on mobile genetic elements they can be freely exchanged between bacteria Chromosomal genes ○ resistance from (rare) spontaneous mutations (usually result in a change in the drug target) R (resistance) plasmids Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 23 Manifest well lezgetit mwah