MICROGENETICS LECTURE - CHAP1&2.pdf
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AYMUNDO, LORENN GLENZ F. R 3MICRO2 Segments of DNA → calledGENES CHAPTER 1.1: Mendel and the Beginning of Genetics Trakr S earch dog that located the final survivor of...
AYMUNDO, LORENN GLENZ F. R 3MICRO2 Segments of DNA → calledGENES CHAPTER 1.1: Mendel and the Beginning of Genetics Trakr S earch dog that located the final survivor of the 9/11 attacks DNA is over6ftlong when unraveled Died via toxic exposure Clonedin 2008 enes- each human has20,000-25,000(this G ○ Via programming DNA to function collectionis called aGENOME) thatprovide instructions for making proteins loningmammals wasunpredictableand C Determine traits (e.g., eye color) or the risk unsuccessful during the early years (2008) of developing diseases ★ Now, it is more successful →raising moral Make up the basicphysicalandfunctional and ethical concernsamong humans units of heredity Within these genes,chemical compounds provide thecodingfor all information → How does cloning work? about a person’sinherited traits Researchers must reprogram an adult Genome- determines a person’s traits by cell’s DNA tofunction like the DNA of an influencing factors on a CELLULAR LEVEL egg enetics- the study ofheredity, expression of G ote:Genes are not the only “factors” that N traits, and the biological inheritanceof traits influence who you are (i.e., environmental between generations factors) Helps us understand thebiological programmingof all life forms EPIGENETICS States that theenvironment and behavior 1 865– Hybridization study of pea plants byGregor can affect the way genes work Mendel Noted the role of“factors”thatinfluence HUMAN GENOME PROJECT the expression of traits → heavily assisted by John Craig Venter ○ Factors = Genes Aims todecode human DNA Identified about99%of the entire human ucleus-storesgenetic information N genetic sequence Chromosomes-carryinformation in the form of deoxyribonucleic acid (DNA) Gregor Mendel Born in 1822 in Moravia (now part of the NA- thehereditary/genetic materialin most D Czech Republic) organisms and carries the genetic information of Son of a tenant farmer said organism Joined a monastery to get an education Adouble helixof nucleotides ○ Where he received the support of ○ Contains aphosphate backbone, Abbot Napp tostudy heredity (in sugar molecules, andnitrogenous peas) bases(Thymine, Adenine, Observed that some pea Cytosine, Guanine) traitsdid not BLEND AYMUNDO, LORENN GLENZ F. R 3MICRO2 S tudied at the University of Vienna from nd Generation 2 1851-1855 (but did not get a degree) Parent:Yellow-seeded plant (self-fertilized) Presented his findings to the Association of Offspring/s:Yellow and green seeds Natural Research in Brno (1865) ○ Few people recognized his findings nalysis:The green trait washiddendue to the A and methods as they were dominant yellow, called the“RECESSIVE”trait uncommon andcontradictory to the BLENDING THEORY(which Conclusion was then widely accepted) Each trait depends on aPAIRof factors (called → ALLELES): ENERAL UNDERSTANDING OF GENETICS G (a) Coming from themother (YY) A. Before Gregor Mendel (b) Coming from thefather (yy) Heredity appearedRANDOM and UNPREDICTABLE Many traits seemed toBLENDin lleles- represent the different variations of a A the offspring(Blending Theory) gene ○ Suggests aliquid factor Heterozygous– different alleles (Yy) controlled heredity Homozygous– same alleles (YY) → Blending Theory of Inheritance Genotype- acombinationof alleles Parental traits mix or blend together Result is called aPHENOTYPE Results in anintermediate offspring ○ E.g., darker skinned parent + lighter How do we visualize how alleles are skinned parent = offspring with a distributed? skin toneIN BETWEEN Inconsistencies are found in traits thatdo V ia thePUNNETT SQUARE not blend away(e.g., red hair) ★ Where thefirst letter of the dominant ○ Persistsinstead of blending from allelewill be used to describe the allele generation to generation distribution (i.e., yellow dominant = Yy) Mendel’s Study of Pea Plants In thefirst generation, each parent gave a Y and → 1 st Generation a y allele. Thus, the offspring areall Parent 1:Purebred yellow-seeded plant HETEROZYGOUS YELLOW (Yy) Parent 2:Purebred green-seeded plant →In thesecond generation, two heterozygous Offspring/s:Yellow-seeded yellow parents will form the following punnett square: nalysis:Mendel called the yellow color trait A “DOMINANT”as it was expressed in all the new seeds hus, offspring possibilities are:HOMOZYGOUS T DOMINANT (YY), HETEROZYGOUS (Yy), and HOMOZYGOUS RECESSIVE (yy) AYMUNDO, LORENN GLENZ F. R 3MICRO2 v irulence factorof the pathogenic strain = DNA is the genetic material production of acapsule P rior to this, the prevailing argument was thatPROTEINS(not nucleic acids) are the → What is the principle of the transformation? carriers of genetic information Uponmixingheat-killed remains of ○ Why proteins? pathogenic strains with living Proteinsaremore complex non-pathogenic strains,SOME BECAME and diversecompared to PATHOGENIC the relatively simpler nucleic ○ Griffith described this as a acid structures TRANSFORMATION(i.e.,a change in genotype AND phenotypedue to The Emergence of Molecular Genetics assimilation offoreign DNA) ○ Competence:the ability of a cell to → One Gene–One Enzyme Hypothesis take up extracellular DNA Main findings:a singlegene controls each environment through stepin the metabolic pathway transportation ○ Concluded that EACH gene controls the production of a specific enzymethat catalyzes a step in the metabolic pathway ○ Most biologists thought that genes werePROTEINS Genes control/regulate specific reactions in the system either by: ○ Acting directly as enzymes, or ○ Determining the specificities of enzymes EXPERIMENTS THAT PROVED DNA TO BE OUR GENETIC MATERIAL Conclusion:R strains transformed into S strains → Evidence that DNA can transform bacteria (heat-killed S cells become incorporated into the genetic material of the R cells,allowing it to code F rederick Griffith for the capsule) Two strains of the bacterium [Streptococcus pneumoniae]were used: (1) pathogenic/smooth, (1) harmless/rough The Avery-McCarty-MacLeod Experiment F irst to announce thatthe TRANSFORMING SUBSTANCE wasDNA ○ Conclusion was based on experimental evidence that only DNA worked in transforming harmless into pathogenic bacterium AYMUNDO, LORENN GLENZ F. R 3MICRO2 T hree setups were used:Proteinase, RNase, and DNase (used toinactivate their respective substrates) ○ Main Finding:Only if DNA is inactivated will the S cellsfail to appear (i.e.,no transformation) Hence, the TRANSFORMING ELEMENT is the DNA Evidence that Viral DNA can program cells A lfred Hershey and Martha Chase R adioactive sulfur and phosphorus(two Viruses (bacteriophages/phages setups) were used to TAG DNA specifically) gave way to strengthen DNA ○ Mixed with host bacteria and being the transforming element centrifuged to separate bacteria ○ Phages:viruses that infect bacteria and phage (m ain structure:protein/lipoprotein Findings:only radioactively head) tagged phosphorus is seen Injects DNAinto the present inside the bacterial bacterial cell to infect them cell How were they certain that it was DNA and → not protein? What is the importance of using sulfur and phosphorus? Sulfur is ONLY FOUND IN PROTEINS Phosphorus is a MAIN COMPONENT OF DNA Experimental design:shows that ONLY ONE of → the two components (either DNA or protein) of a phage known as T2 enters anE. coliduring infection AYMUNDO, LORENN GLENZ F. R 3MICRO2 The Discovery of DNA (video notes) Discovery of the DNA Structure T he three dimensional arrangement of Early concept:Chargaff’s Rules atoms in biological molecules (responsible ○ Two double-helix strands areheld for genetic information)shouldexplain the together by HYDROGEN BONDS stabilityof life AND themutabilityof life BETWEEN NUCLEOTIDE BASES ○ Stability:so that traits can be ○ Bases of the two DNA strands in a passed down from generation to double helixpair in a consistent generation way(i.e., A-T and G-C) ○ Mutability:have change so that WhereG-C contentcan be evolution can occur used for classification James Watson and Francis Crick ○ Proportions of A and G vary among Chromosomes:made up ofproteins and species nucleic acids (DNA) X-ray crystallography Rosalind Franklin’s X-ray diffraction image of ○ A powerful technique forsolving DNA (Photo 51) molecular structure ○ Can determine thepositionof every single atom in the molecule ○ Resulting picture is adiffraction pattern Pauling, Watson, and Crick suggested that DNA must be ahelixof some kind Franklin was an X-ray crystallographer (took the → CHAPTER 1.2: Genetics and Genetic Elements image “Photo 51”) → the image was thefinal piece of the puzzle needed to determine the structure of DNA enetics and Genetic Elements G Deoxyribonucleic acid (DNA) atson, Crick and Wilkins W Thebackboneof DNA chain is alternating → discovered the STRUCTURE of DNA phosphates and pentose sugar → findings were based on theprinciples of (deoxyribose) Chargaffand theX-ray crystallography images of Phosphodiester bonds connect3’-carbon Franklin of one sugar to5’-carbon → awarded with a Nobel Prize in Physiology and Double helix (two strands) with an Medicine in 1962 antiparallelconfiguration (5’- to 3’-and3’- Franklin was not a recipient due to her to 5’) deathin 1958 ○ 5’- has aphosphategroup ○ 3’-hydroxylgroup (—OH) Counting the 5’- and 3’- ends must start CLOCKWISE from the oxygen DNA size is expressed bynumber of BASE PAIRS ○ 1000bp = 1Kb Mosteukaryotes→ linear DNA configuration AYMUNDO, LORENN GLENZ F. R 3MICRO2 → How are supercoils inserted and removed? Facilitated by enzymes called Topoisomerases ○ enzyme thatfacilitates supercoiling DNA gyrase Typeof topoisomerase Introduces abreakin the supercoiled DNA for a small section ○ Introduces atwistand rejoins it Most common topoisomerasein bacteria DNA IN BACTERIA and most archaea responsible for Supercoiled (hasmultipleturns) supercoiling ○ For it to fitinsidethe cell ○ Usually found inprokaryotes Chromosomes Double-strandedCIRCULARDNA DNA wraps around“spools” of proteins ○ As opposed to linear DNA in calledHISTONESthatallow chromosomes eukaryotes to pack tightly together E. colihas around5 mega bp ○ Histonesare only found in ○ Genomes are quite big → when eukaryotes and archaea lined out, the length wouldexceed ○ In bacteria = NO HISTONES the cell size Each DNA molecules consists oftwo For the genome to fit inside, strandstwisted into adouble helix bacteria muststrategizeto In cells,DNAmolecules and their pack the DNA(i.e., associatedproteinsareorganized into supercoiling) chromosomes DNAdirectionby which it coils can determine if it ispositiveornegative Positive vs Negative Supercoiling Positive Supercoiling Negative Supercoiling Has more stress (more Much morerelaxed pressureapplied) Facilitatesunwinding Harderto unwind for replication and Present in MOST transcription archaea Present in majority of prokaryotes&bacteria (also somearchaea) Extremophilic archaeans When a cell prepares todivide, itduplicates its → Can survive inextreme conditions(e.g., entire chromosome,forming theSISTER extreme temperature) CHROMATIDS Haspositive supercoilingthat provides → When the cell divides, both daughter cells have thermal stabilityand are harder to theexact same copiesof the chromosomes denature AYMUNDO, LORENN GLENZ F. R 3MICRO2 Sister chromatids Chromosome number ○ one of the two attached members Aeukaryoticcell’s dna is divided into a of a duplicated eukaryotic characteristic number of chromosomes chromosome Thenumber of chromosomes presentin Centromere a eukaryotic cell ○ constricted regionin a eukaryotic ○ Thesum of all chromosomesin a chromosomewhere sister cell of a given type chromatids are attached ○ Ahumanbody cell (diploid) has46 chromosomes ○ Diploid:cells havingtwo of each type of chromosomecharacteristic of the species(2n) Means there are a total of23 pairsof chromosomes → Thenumber of chromosomesdo not describe the complexityof organisms Trisomy A condition that bears anextra duplicated LINEAR chromosome A chromosome most bacterial chromosomes arecircular ○ Having3 instead of a pair (not linear) Most cases are FATAL ○ Most result in miscarriage HOW DNA CONDENSES/FORMS A STRUCTURE Trisomy 21:survivabletype of trisomy, 1. Starts withDNA strand(double-stranded) (better known asDown syndrome) 2. At regular intervals, the DNA strand will WRAP ITSELF into proteins(histones) ○ Histones(ball-like structure)pick Eukaryotic Chromosomes (2 Types) upDNA strands like a thread and Autosomes spoolthe DNA around the protein ○ Paired chromosomeswith the 3. Histones with spooled DNAtwist together samelength, shape, centromere to formfiber-like structures location, and genes 4. These “fibers”coilagain into ahollow ○ Any chromosomeother than a sex cylinderto form achromosome chromosome ○ In humans,22 of 23pairs are AUTOSOMES (the remaining ONE is asex chromosome) Sex chromosomes ○ Members of a pair of chromosomes thatdiffer between males and females ○ TheLASTpair ○ In humans,XYandXX → NOTE:Same chromosomes (XX) ≠ always FEMALE (it is only applicable in humans) AYMUNDO, LORENN GLENZ F. R 3MICRO2 Karyotyping ○ U nlike chromosome number, the Reveals characteristics of an individual's GENOME SIZEdescribes the chromosomes complexityof organisms Visualizesan organism’s chromosomes Biggergenome =more Karyotype complex ○ Image of an individual’s E.g. prokaryotes have complement of chromosomes 10-15mbp, whereas viruses arranged by size, length, shape, and (more simple) have around centromere location 2kbp-1mbp Smallestgenome of bacteria recorded is 159kbp Discovery ofGIANT viruses = have around3mbp Some (not all) genes encoding enzymes of a single biochemical pathway are clusteredintoOPERONS, transcribed to form a single mRNA, and regulated as a unit Other genes of biochemical pathways are not clustered ○ They aredistributedall throughout the genome (most are spread out) Bacterial Chromosomes Operonsare mere C hromosomes:main genetic elementin exceptions prokaryotes Other (nonchromosomal) genetic elements include: ○ Virus genomes ○ Extrachromosomal DNA (plasmids) ○ Organellar genomes Mitochondrial DNA ○ Transposable elements Most bacteria and archaea have aSINGLE CIRCULAR CHROMOSOME ○ Eukaryoteshave 2 or more LINEAR chromosomes Escherichia coli Genome size:has around5 mega bp (mbp) In the 5mbp, there are almost4300 possibleprotein-encoding genes ○ Make up88% of the genome(not all DNA code for protein) Compactrelative to eukaryotes AYMUNDO, LORENN GLENZ F. R 3MICRO2 lac Operon Baltimore Classification Scheme Most well-knownoperon Groupsviruses depending on theirgenetic Facilitates thebreakdown of lactose material mRNA istranscribed as a unit 7 classifications(numbered 1 to 7) 7 Classifications of Viruses According to BCS ouble-strandedDNAviruses D 1 (e.g., adenoviruses, herpes viruses or HSV1 & 2, chickenpox, etc.) ingle-strandedDNAviruses S 2 (e.g., parvoviruses) Prokaryotic vs Eukaryotic Chromosomes ouble-strandedRNAviruses D 3 (.e.g., gastrointestinal diseases) ingle-strandedRNA positivesense S 4 (e.g., picornaviruses, coronaviruses) ingle-strandedRNA negativesense S 5 (e.g., orthomixoviruses) ingle-strandedRNAwithreverse S 6 transcription ouble-strandedRNAwithreverse D 7 OTHER GENETIC ELEMENTS transcription RT-PCR Gold standard for theidentification of COVID-19 “RT”meansreverse transcription V irusescontaineitherRNA or DNA ○ Reverse transcription:where RNA genomes(never both) converts back into DNAinstead of ○ Can be linear or circular proteins Vast majority are LINEAR RNA→DNA→RNA→Protein ○ Can be single or double-stranded Example: retroviruses, HIV In viruses,we pay more attention to:(if DNA or RNA) and (if single or Why does RT-PCR detect coronaviruses if it → double-stranded) does not undergo reverse transcription? PCR makes multiple copies of fragment DNA ○ Coronaviruses have RNA → in order to make multiple copies of the viral genome →RNA must be CONVERTED TO DNA Hence,reverse transcription AYMUNDO, LORENN GLENZ F. R 3MICRO2 P CRcannot denaturea single-stranded Initially, the science community labeled DNA as → RNA aSTABLE (fixed) molecule Transposable Elements Barbara McClintock segments of DNA thatcan move from one - Responsible for the discovery ofjumping site to another siteon thesameor genes differentDNA molecule - Study on corn (kernel) colors Inserted intoother DNA molecules: - Awarded in 1983 (won asoloprice for chromosomes, plasmids, viral genomes Physiology and Medicine) Also calledjumping genesortransposons Can be placed (theoretically) in ANY PART TRANSPOSABLE ELEMENTS SELF-REPLICATE of the genome THROUGH TWO MAIN MECHANISMS ○ Depending on the placement, the transposon can become nonfunctional (or no effect) There arecertain places where it becomes effective or beneficial Donor DNA The DNA that JUMPS from one place to another LASS 1 C → The donor DNA istranscribed into an RNA intermediate → Itreverse transcribesBACK into aDNA intermediate → The DNA intermediateintegratesinto the other (target) section of DNA Like acopy + paste CLASS 2 simpler (easier) →Excisionof the transposon occursDIRECTLY and is immediatelyintegratedinto the target DNA section Like acut + paste AYMUNDO, LORENN GLENZ F. R 3MICRO2 INSERTION SEQUENCES ○ S ome cells containMULTIPLE DIFFERENT plasmids Extrachromosomal DNA(not part of the chromosome) Double-stranded DNA thatreplicates separatelyfrom chromosomes Usuallycircular Generallybeneficialfor the cell (e.g. antibiotic-resistance) Can betransferred directly ○ Usually through CONJUGATION (or horizontal gene transfer) Not extracellular, unlike viruses There are thousands of plasmids currently known ○ E.g.,E. colihas 200-300 plasmids identified A type of transposon thatconfers antibiotic-resistance genes → Inverted vs Direct repeats GENETIC ELEMENTS: CHROMOSOMES AND PLASMIDS Plasmids F ound in manybacteria and archaea Genetic information encoded on plasmids isnot essential for cell functionunder all conditions ○ BUT itmight give advantagesto the cell May confer aselective growth advantage under certain conditions Range in size from 1kbp to more than 1mbp ○ Smallercompared to DNA AYMUNDO, LORENN GLENZ F. R 3MICRO2 R hizobiarequires plasmid-encoded functions tofix nitrogen Metabolism (hydrocarbon degradation) Important forconjugation(horizontal gene transfer) DOLLY THE SHEEP First successfully cloned mammal R plasmids Resistanceplasmids Confer resistanceto antibiotics or other growth inhibitors A widespread and well-studied group of plasmids → How was Dolly’s cloning done? Severalantibiotic resistance genes can be A pipette is used toremove the nucleus onone R plasmid from an egg cell e.g. Plasmid R100 ○ A nucleus is thenimplantedfrom a DONOR CELLinto the sample cell (surrogate) Example:A nucleus from Organism A’s egg cell is replaced with the nucleus from Organism B. The egg cell is then implanted into Organism C. Genetically, the result will be a copy/clone of Organism B(due to the genetic information transferred from Organism B). → In humans, PARTIAL CLONING is more accepted Partial cloning ○ Synthesize organs ○ Uses somatic stem cells THER BENEFITS OF PLASMIDS O THE RISE OF ANTIBIOTIC RESISTANCE In several pathogenic bacteria,virulence Penicillinand other B-lactam antibiotics factors(ability to attach or produce toxins) act byinhibiting penicillin-binding areencoded by plasmid genes proteins, whichnormally catalyze Bacteriocinscan be encoded on plasmids cross-linking of bacterial cell walls ○ Cankill or inhibitclosely related ○ They “kidnap” or take awaythe groups of bacteria(competition for penicillin-binding proteins nutrients) ○ So that it will not help build the cell ○ Produced by some bacteria wall(cell wall synthesis is disrupted) AYMUNDO, LORENN GLENZ F. R 3MICRO2 R esistance to penicillin viamodified CHAPTER 2: The Flow of Genetic Information cross-linking enzyme Antibiotic resistance canmis-spreadvia plasmids IMPORTANT MACROMOLECULES 4 ○ Can transfer from one bacteria to Proteins another viahorizontal gene Lipids(non-informational) transfer Carbohydrates(non-informational) Nucleic acids → Lipids and Carbohydrates Important instructuralcomponents of the cell,compositionof the cell membrane, or metabolic processes withno information → HOW DOES ANTIBIOTIC RESISTANCE OCCUR? 2 Informational Macromolecules Proteins ○ Built on by amino acids Nucleic acids ○ Two types →DNA and RNA → These two are responsible for bringing in the information that encodes the phenotype(or characteristics) of the organism → Without these two, there would be no TRAITS How do we create these materials → (informational macromolecules)? Usingthree important stepscollectively called theFLOW OF GENETIC INFORMATION: ○ Replication ○ Transcription ○ Translation Also called the“Central Dogma” AYMUNDO, LORENN GLENZ F. R 3MICRO2 TRANSCRIPTION D NA to RNA Main enzyme:RNA polymerase TRANSLATION RNA to protein ○ Proteins fulfill all thechemical processes Main enzyme:Ribosomes In viruses (despite being noncellular organisms), → follow the same processes(although some VIOLATE the central dogma) E.g. inRNA-containing viruses,reverse transcriptionis usually performed as the THE CENTRAL DOGMA first step GENETIC INFORMATION FLOW: EUKARYOTES VS. PROKARYOTES . G A ENERAL DISTINCTION Eukaryotes ○ Presenceof nucleus ○ Multicellular (and unicellular) ○ Membrane-boundorganelles ○ Divide bymitosis(mitotic division) Reproduce asexually or sexually Prokaryotes ○ Absenceof nucleus ○ Unicellular REPLICATION Some are capable of H appens when the DNA (information formingfilaments carrier)replicates into two sets ○ Notmembrane-bound ○ Distributedinto the2 daughter ○ Divide bybinary fission(in the case cells of bacteria) When the cell performs its metabolic Usually reproduce in a set of processes, thematerials needed are colonies (?) encoded in the DNA(to be transcribed) Main enzyme:DNA Polymerase AYMUNDO, LORENN GLENZ F. R 3MICRO2 . G B ENETIC INFORMATION FLOW Why are eukaryotic genes transcribed → Eukaryotes individually and not in clusters (unlike ○ Each gene istranscribed prokaryotes)? INDIVIDUALLYinto asingle mRNA InEukaryotes,replication and ○ Replication and transcription occur transcriptionarehappening INSIDE the in theNUCLEUS nucleus ○ RNAs must beexported outside ○ Whereastranslation occurs nucleusfortranslation OUTSIDEthe nucleus (i.e. into the Prokaryotes cytoplasm, particularly in the ○ Multiple genesmay be transcribed ENDOPLASMIC RETICULUMfor the inone mRNA ribosomes) ○ Coupledtranscriptionand ○ Hence, theycannotoccur translationoccur (happens at the SIMULTANEOUSLY same time) Producing proteins at I nProkaryotes, as theyDO NOT have a MAXIMAL RATE (faster) nucleus ○ Thethree processescan bemade simultaneously ○ Coupledtranscription and translation can occur Becausethey occur within the cytoplasm(for prokaryotes) Why do we transcribe eukaryotic genes only → ONE GENE AT A TIME? Because ofintrons(i.e. noncoding regions) present in between/in the DNA or gene of eukaryotes (or the extrons) ○ Prokaryotesdo not have these noncoding regions, hence,multiple genes can be ONE AFTER ANOTHER AYMUNDO, LORENN GLENZ F. R 3MICRO2 NA REPLICATION: D Copying the Genetic Blueprint I t is important when two daughter cells divide, they willcarry BOTH of the genetic materialpresent in the mother cell ○ This process is called the REPLICATION Important Concepts: DNA Template ○ Precursorof each nucleotide is a deoxynucleotide 5’-tripophosphate (dNTP) [VIDEO NOTES] DNA REPLICATION In order topass genetic informationon to its offspring, an organism mustmake a copy of its DNA(i.e. replication) During replication, each strand of the NOTE: Uracilonly plays a role inRNA parentalDNA serves as atemplatein the creation of new DNA → DNA replication is semiconservative Since each newly synthesized DNA is made up of only1 parentalstrand and1 newstrand,DNA REPLICATION IS DESCRIBED AS SEMICONSERVATIVE ○ Semiconservative:1 strand in each molecule is conserved, while the other is newly replicated ENZYMES INVOLVED IN DNA REPLICATION DNA Polymerase W hen the DNA replicates, it synthesizes Catalyze polymerizationof new copies of DNA deoxynucleotides ○ Usesboth strands as templates Primaryenzyme (replicates DNA) ○ When a cell divides, it carriesone In E. coli, there are FIVE different DNA oldstrand andone newly polymerases replicated ○ DNA Pol I:playslesser role Replication proceeds ONLY from the5’ ○ DNA Pol II:repair damage end to the 3’ end ○ DNA Pol III:primary enzyme replicating chromosomal DNA ○ DNA Pol IV:repair damage ○ DNA Pol V:repair damage AYMUNDO, LORENN GLENZ F. R 3MICRO2 I t isimportantto replicate DNA with THE PROCESS OF DNA SYNTHESIS extraordinary fidelitybecause this ensures there areNO GENE MUTATIONS 1. D NA synthesis begins at theORIGIN OF ○ Gene mutationscan cause REPLICATIONinprokaryotes irregularitiesandcomplicationsin Origin of replication:middle the organism ○ Aportionwithin the chromosome Rendering the genes where replication is INITIATED uselessordifferent ○ Where thereplisomesmove to Highlights the importance Replisomes:responsible for ofaccuracyin the replicating DNA replication process 2. The DNA helix in the origin is initially ○ Mistakes can also lead to achange OVERLAPPED in the phenotype 3. Proteins(helicase) willopenthis up to create a single-stranded DNA (allowing → Why is DNA supercoiled? polymerases to make copies of the parent Tomaximize the spaceinside the cell strand) (packing) ○ Allows the DNA to becompacted withinthe cell This supercoiling is removed (relaxed) when undergoingreplication GENES AND THE ENZYMES THEY CODE Replicationis done atBOTH strandsof DNA (as → templates) Differentiatesreplication with transcriptionwhere only ONE STRAND of RNA serves as a template AYMUNDO, LORENN GLENZ F. R 3MICRO2 DNA helicase DNA Polymerase III The enzyme thatunwinds or opens upthe Adds1000 nucleotides per second DNA double helix ○ Very quick ○ Creates thereplication fork Replication fork:zone of ORDER OF ENZYME ACTIVITY: unwoundDNAwhere 1. U NWINDING:Helicasecuts dsDNA replication occurs 2. Ssbpbinds to single-stranded DNA a. Ensures that they willnotrewind back 3. Replisomesfollow → REPLISOME:complex enzyme containing the primase (that creates a primer) and the polymerases 4. Primaseattaches to ssDNA and creates an ydrogen bonds:bonds connecting nucleotides H RNA primer in the DNA 5. DNA Polymerase IIIsynthesizes the Weak bond; can easily be CUT and majority of the DNA sequence connected again a. Moves it to thereplication fork What the helicase cuts down 6. DNA Polymerase Iremoves the RNA primer and replaces it with DNA Single-stranded binding protein (Ssbp) a. Then in thelagging strand,DNA Stabilizessingle-stranded DNA so it will Ligaseseals the cut by connecting not BIND BACK(since it can easily fragments after primer removal(to connect) complete the sequence) ○ Allows primase to be attached to create theprimer Primer ashort stretchof RNA ○ Made fromRNAbyprimase ○ Located at the initiation of DNA synthesis Once it is attached, it → NOTE:DNA isantiparallel synthesizes the component One has a direction of 5’ to 3’ of the DNA One has a direction of 3’ to 5’ EXTENSION OF DNA O ccurscontinuouslyon theleading strand ○ The one having a5’ to 3’ direction Occursdiscontinuouslyon thelagging strand (No3’ –OH) OMPLIMENTARY BASE PAIRING C ○ Lagging strand: Adenine-Thymine A thousand bases are Cytosine-Guanine neededbefore another primer is attached AYMUNDO, LORENN GLENZ F. R 3MICRO2 C ontainfragmentsof DNA APPLICATION: Needs to be sequenced in If an E. coli has 4.6mbp, how long (in theOPPOSITE DIRECTION minutes) will it take a DNA polymerase to complete replication? (Note: DNA pol III adds 1000 nucleotides per second) Connecting DNA fragments on the lagging → 4. 6𝑚 𝑏𝑝 = 4, 600, 000𝑏𝑝 strand = 4,600,000𝑏𝑝 1000 DNA Ligase seals the nicks in the DNA (i.e. 4,600𝑏𝑝 = 60𝑠 connects the fragments) = 76𝑚 𝑖𝑛𝑢𝑡𝑒𝑠𝑡𝑜𝑡𝑎𝑙 = 3 8𝑚 𝑖𝑛𝑢𝑡𝑒𝑠𝑝𝑒𝑟𝑠𝑖𝑑𝑒( 𝑏𝑖𝑑𝑖𝑟𝑒𝑐𝑡𝑖𝑜𝑛𝑎𝑙) Replisome Acomplexof multipleproteinsinvolved in replication ○ Enzymes used in replicationMOVE AS ONE as the replisome Because DNA isflexible, the leading and lagging strands are replicated simultaneously BIDIRECTIONAL REPLICATION → DNA synthesis isbidirectional Inprokaryotes, it is because they have a CIRCULAR chromosome → Bidirectional synthesis involves two replication forks moving inopposite directions Both can perform DNA synthesis AYMUNDO, LORENN GLENZ F. R 3MICRO2 DNA MISMATCH REPAIR MECHANISM TRANSCRIPTION PROCESS IS DIVIDED INTO THREE MAJOR STEPS: INITIATION, ELONGATION, TERMINATION INITIATION Involvestwoimportant enzymes/proteins: ○ RNA polymerase Responsible for transcribing atch supplementary video: W DNA to mRNA https://www.youtube.com/watch?v=BGzz712Z0n ○ Sigma factor 8&t=344s Identifies thepromoter region Tells thedirectionby which the transcription process RNA SYNTHESIS: TRANSCRIPTION should proceed i.e., if the primer TRANSCRIPTION region is at the RNA Synthesis OPPOSITE strand → Carried out byRNA Polymerase(primary direction will enzyme) proceed at its other RNA polymeraseuses DNA as a template strand ○ Inreplication, template is TWO → Thesigma factorwill bring in RNA polymerase STRANDS into the regionwhere you want to transcribe it ○ Intranscription, template used is Once they arebound, the sigma factor will ONE STRAND bereleased(and RNA polymerase will start What will happen if we use the transcription) BOTH strands of the template?:Differencein the amino acid sequence Precursors (materials) needed to make RNA include: ○ dNTPs →ATP,GT P,CTP, andUT P Different from replication dNTPs only by the replacement of THYMINE ○ Movement of the chain growth is from the5’ TO 3’similar to DNA replication It should have a3’ to 5’ direction for thetemplate Only 1 strandis transcribed Unlike replication,no priming needed ○ Primers are not necessary AYMUNDO, LORENN GLENZ F. R 3MICRO2 ELONGATION ○ - 35 region:35 basesbeforethe RNA polymerasemakes copiesof DNA start ○ But instead of using DNA, it uses This problem is not experienced with RNA(A-U & G-C) primers → we candifferentiate the RNA from the actual sequence included TERMINATION Triggered when the process reaches a Only 1 sigma factor is needed to recognize → certain point where theRNA process will different sequences be told to stop If each sequence needs different sigma ○ RNA will bereleasedand factors, you would need one SF for one translation can be performed gene(not ideal) RNA polymerase and the Promoter sequence Consensus region:an area where certain → RNA polymerasehas5 different subunits conserved sequences would berecognized by the Thesigma factorof RNA polymerase SF recognizes initiation siteson DNA called PROMOTERS ○ Pribnow box(–10 region) and TTGACA(–35 region) How does polymerase differ from bacteria to → archaea to eukarya? ARCHAEA:13 subunits How does a sigma factor know when a → sequence is the promoter region? By recognizing aPRIBNOW BOX(-10 region) ○ -10 region:10 basesbeforethe start of transcription ○ Also called aTATA boxbecause it has a TATA sequence ○ Corresponds toAUGor Methionine By recognizing aTTGACA(-35 region) AYMUNDO, LORENN GLENZ F. R 3MICRO2 Different sigma factors have different → T hree types of rRNA:16s, 23s, and 5s + recognition sequences tRNA → How are bacterial cells transcribed? Would it be possible for the RNA polymerase → to continuously transcribe the whole set? Yes, but not ideal Termination is REQUIREDbecause a certain product of transcription 1. D NAcarries the information needed corresponds to a certain protein (specific 2. DNA is transcribed to aprimary transcript amino acids) 3. Segments called‘intervening spaces’ ○ Transcription should only produce should beremovedbefore making the theproteinsthat we NEED primary transcript Production of unnecessary a. Do not code for anything proteins would mean b. Calledjumping genes constant utilizationof c. Has to be removed from the resources sequence 4. The primary transcript willcorrespond to TRANSCRIPTION IN BACTERIA the RNAs involvedin thetranslation 8 Transcriptional units:DNA segments process transcribed into 1 RNA moleculebounded byinitiationandterminationsites Most genes encode proteins, but in some genes,RNAs are not translated into proteins ○ These are the genes that will encode for the ribosomes(e.g., rRNA, tRNA) First transcribed into mRNA (?), thenpackagedto create tRNA and rRNA AYMUNDO, LORENN GLENZ F. R 3MICRO2 I n bacteria, genes are transcribed into OPERONS(i.e. clusters of genes → different genes correspond to different enzymes) Onlyone promoter regionis responsible for regulating the expression of genes ○ Example:genes for regulation of lactose (three enzymes degrade lactose → all three are encoded by one promoter region) Operons are transcribed into asingle mRNAcalled apolycistronic mRNA containing multiple open reading frames that encode amino acids How do we know if the area or region of the → DNA is the TERMINATION SITE? Governed by specific DNA sequences ○ E.g.GC-rich sequencewith inverted repeat and central nonrepeating segment TRANSCRIPTION IN ARCHAEA AND Rho-independent termination:RNA EUKARYA polymerase recognizes the sequence, and A rchaeal and eukaryotic RNA polymerases, asignal is sentfor the RNA polymerase to promoters, and terminators dissociate and stop transcribing ○ Similar,more complexthan ○ Requires a lot of GC sequences bacterial RNA polymerases ○ Creates a stem-loop structure ArchaeacontainoneRNA polymerase (secondary structure for RNA) ○ Resembleseukaryotic polymerase II AsRNA can only be a single ○ Eukaryoteshave3 RNA strand, this loop signals the polymerases RNA polymerase toendthe transcription RECOGNITION SITES: TATA BOX Rho-dependent termination:Rhoprotein reognizes specific these DNA sequences andcauses a PAUSEin the RNA polymerase ○ ReleasingRNA and RNA polymerase ○ Theproteinsignals the polymerase to stop transcription Same concept as thebacterialTATA box ○ One regionrecognized by the polymerases→startstranscription AYMUNDO, LORENN GLENZ F. R 3MICRO2 NOTHER MAJOR DIFFERENCE IN A EUKARYOTIC TRANSCRIPTION IS CODING AND NONCODING GENES Eukaryoticgenes havecodingand noncodingregions ○ Exons:Codingsequences ○ Introns:InterveningNONcoding sequences Very rarein archaea Found in tRNA and rRNA encoding transcripts Removed byspecial ribonuclease ○ RNA processing is required to form mature RNAs for translation Large regions calledNONCODINGregions MUST BE REMOVEDbefore transcription Unlikebacteria and archaea, where all genes are already there ○ Coding spaces areeasily removed What will happen if noncoding regions are → not removed? If a protein requires 10 amino acids, and a coding region corresponds to two amino acids in between, at the end → product will have 12 amino acids ○ Not accurate,as only 10 are needed Different amino acids → different protein structure (thischangetherefore induces animpacton the protein product’sproperties) Joined exon products proceed to translation, → RNA processing in Eukaryotes and intervening and the intron is degraded(spliceosome is sequences in Archaea recycledby the cell) Splicing:the process ofcutting off noncodingregions to fuse together the M ature mRNA:only has thegenes needed coding regions and has totravelfrom the nucleus into the ○ Removingintrons (in between) and cytoplasm (where translation occurs) joiningexons In eukaryotes:splicing occurs in the nucleusvia theenzymespliceosome (RNA+protein) AYMUNDO, LORENN GLENZ F. R 3MICRO2 Where do we find RNA and DNA insi
