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69 Cards in this Set
- Front
- Back
Key processes involving genetic information: |
-synthesis of the three types of informational macromolecules 1. Replication - making a copy of the DNA 2. Transcription - synthesis of RNA from DNA template 3. Translation - synthesis of proteins from mRNA |
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Linear Chromosomes |
Eukaryotic chromosomes are linear. Some viral chromosomes are also linear. |
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Escherichia coli K-12 chromosome |
-4, 639, 221 base pairs - 4288 ORFs - Single origin of replication – oriC– a tandem array of 3 nearly identical nucleotide sequences and 4 DNA proteinbinding sites. - Five copies of the transposable element IS3 are in blue -Site where bacteriophage lambda integrates is shown in red -The genes of the maltose regulon (transcriptionally controlled by the same activator) which includes 2 maltoseoperons and malS arein green. |
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Genetic map of the resistanceplasmid R100 |
A conjugative plasmid capable oftransfer between enteric Gram-negative bacteria like Escherichia, Klebsiella, Proteus, Salmonella, andShigella. mer –mercuric ion resistance sul –sulfonamide resistance str –streptomycin resistance cat – chloramphenicol resistance tet– tetracycline resistance oriT– origin of conjugative transfer IS10 – insertion sequences for thetransposable element Tn10. |
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Structure of DNA |
•2polynucleotide strands run antiparallel •strands have complementary sequences •hydrogen bonding Adenine = Thymine (A=T) Guanine = Cytosine (G=C; 3 hyrdogenbonds) |
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TEM of an E. coli cell(large white) that was gently lysed to allow the highly compacted DNA (spaghetti looking stuff) to be released. White arrows are pointing to the much smaller plasmids. |
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Chromosomes by Domains |
Most Archaeasupercoil their DNA with DNA gyrase,but some species use histones to form nucleosomes |
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DNA by Domain |
The largestplasmids known are found in the Archaeaextreme halophile species Halobacterium and Halococcus |
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Types of RNA |
• messenger RNA (mRNA) – translated into protein • transfer RNA (tRNA) – carry amino acids to be put into proteins • ribosomal RNA (rRNA) – brings together mRNA and tRNAs for proteinsynthesis |
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Transcription |
• mRNA,tRNA, and rRNA are transcribed from DNA • promoter – DNA sequence upstream of transcriptionstart site • transcription start site – deoxyribonucleotide where RNApolymerase starts transcription • RNA polymerase – adds ribonucleotides complementary to the DNA template: A–T– G – C U–A– C – G forminga single stranded RNA molecule • terminator – sequence where transcription stops |
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Transcription in Eukaryotes |
-Asingle gene is transcribed at a time in eukaryotes -Occurs in the nucleus -3 RNA polymerases I – rRNA II – mRNA III - tRNA |
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Noncoding DNA sequences |
intron |
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Coding DNA sequences |
exons |
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Transcriptionfactors |
-help RNA polymerase bind to promoter (no sigma factor) |
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Transcription in Archaea |
-thepromoter for a gene in Archaea is similar to eukaryote promoters -TBP and TFB bind to DNA -RNA polymerase bind to promoter region and begins transcription -Nointrons in prokaryote genes. |
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Transcription in Bacteria |
•singleRNA polymerase with a sigma factor attached •sigmafactor binds to promoter then RNA polymerase starts transcription |
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Co-transcription |
-only occurs in prokaryotes -no introns in prokaryote genes -multiple genes can be transcribed with the same RNA polymerase -forms a polycistronic mRNA |
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Terminationof Transcription |
•Rho-dependent termination •Intrinsic terminators |
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Rho-dependent termination |
•Rho-dependent termination –Named after the E. coli protein Rho –Rho binds to RNA moving toward RNA polymerase - DNA complex; –Rho removes RNA polymerase when RNA polymerase reaches the Rho-dependent termination site **Archaea and Eukaryotes do not have Rho-dependent termination. |
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Intrinsic terminators |
–Noextra proteins required –Ex.Inverted repeats in DNA sequence forming stem-loop structure aftertranscription |
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Inverted Repeats |
-Instabilityof the temporary A:U base pair between the adenines in the DNA template and theuracil’s in the RNA transcript causes RNA polymerase to pause and transcriptionto terminate |
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Transcription Comparison |
-Archaea has more transcription factorsthan Eukarya -SeveraltRNA and rRNAencoding genes of Archaea have introns that are splicedout -These archaealintrons are excised by a specific endoribonuclease thatrecognizes exon-intron junctions |
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Operon |
•several genes co-transcribed •encode proteins or rRNA thatare used together •operonsare only found in bacteria and archaea •operonsare found in chloroplast and mitochondrial genomes of eukaryotes |
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Regulation of Transcription |
-similar in bacteria and archaea -eukarya has additional regulatory controls Positive vs. Negative |
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Negative Control of Transcription |
• Prevents transcription • Involves – Enzyme repression – Enzyme induction |
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Enzyme Repression |
-occurs when sufficient product is present to stop synthesis of enzymes nolonger needed |
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arg Operon |
-in the absence of arginine, the repressoris not bound to the operator -in the presence of arginine, argininebinds to the repressor allowing the repressor to bind to the operator to blocktranscription |
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Proteins Coded by the 3 genes in arg Operon |
Theproteins coded for by the 3 genes in the argoperon are enzymes needed to make arginine: • argC –N-acetyl-gamma-glutamyl-phosphatereductase • argB – acetylglutamatekinase • argH – argininosuccinate lyase |
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EnzymeInduction |
occurs when substrate is present to make enzymes needed to use substrate. |
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lac Operon |
-in the absence of lactose, the lac repressor binds to the lacoperator to block transcription -in the presence of lactose,the lac repressor binds allolactose (theinducer) thereby removing the lac repressor from the lacoperator and allowing transcription. (See cataboliterepression: glucose must also be absent for lacoperon induction.) |
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Genes Coded by lac Operon |
• lacZ encodes beta-galactosidase that cleaves lactose into glucose and galactose • lacY encodes a permease that transports lactose into the cell • lacA encodes a beta-galactoside transacetylase acetylates lactose; function unclear *Delection of lacY or lacZ yields lac- phenotype; cells are unable to use lactose. |
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Positive Control of Transcription |
•Activation of transcription •Controlled by binding of activator protein when inducer is present |
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Catabolite repression |
•repression by catabolite instead of repressor protein •“glucose effect” •Diauxic growth •cells will always use glucose first |
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Diauxic growth |
-the diphasic response of a culture of microorganisms based on aphenotypic adaptation to the addition of a second substrate -characterized by agrowth phase followed by a lag after which growth is resumed |
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Overallregulation of the lac system |
1. In the absence of glucose, there issufficient cAMP tobind to CRP (cyclic AMP receptor protein) -positive transcriptional control 2. When lactose is present, lactose is takeninto the cell and converted to allolactose -negative transcriptional control • conditions1 and 2 must be met for induction of the lacoperon or there is no induction |
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CRP |
(cyclic AMP receptor protein) an activator protein and when bound by cAMP can bind to the activator binding site (labeled C and highlighted blue in the figure) in the promotor of the lac operon |
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allolactose |
binds to the lac repressor removing the lac repressor from the lac operator |
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lacoperon repression by glucose |
•Glucoseinhibits cAMPsynthesis - If there is not sufficient glucose then cAMPwon’t bind to the CRP protein and CRP won't bind to the CRP-binding site •RNApolymerase will only bind to promoter if CRP protein is bound to CRP-bindingsite *Glucoserepresses the transcription of other genes and operons in addition to the lacoperon in a similar manner |
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maloperon |
-in the absence of maltose, there is noactivation of the mal operon -in the presence of maltose (inducer), maltose binds to the maltoseactivator protein allowing the maltose activator protein to bind to theactivator binding site to activate transcription. -positive control |
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proteins coded by genes on mal operon |
-the proteins coded for by the genes of the mal operon are required to bring maltose into the cell so maltose can be broken down by other enzymes to be used as an energy source. -maltose is a disaccharide of glucose. MalE – periplasmic maltose binding protein MalF – maltose transporter membrane protein MalG – maltose transporter permease |
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Regulon |
–more than one operon under the control of a single regulatory protein -thegenes of the maltose regulon(transcriptionally controlled by the same activator) which includes 2 maltoseoperons and malS are in green |
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arginine regulon |
arginine biosynthetic enzymes are all under the control of the arginine repressor protein |
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Controlof Transcription in Archaea |
-uses repessorproteins like NrpR -NrpRrepresses genes active in nitrogen assimilation, such as genes needed fornitrogen fixation and glutamine synthesis -when organic nitrogen is plentiful, NrpR represses by binding to thepromoter of various nitrogen assimilation genes -when nitrogen becomes limiting (indicatedby rising levels of α-ketoglutarate) and not enough ammonia is available, α-ketoglutarate willbind to NrpR torelease NrpR fromthe promoter and allow transcription of nitrogen assimilation genes *activator proteins also regulate transcription in Archaea. |
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Regulation of Differentiation at the level of transcription |
-2 componentregulatory system -thesensor kinase detects the environmental signal and autophosphorylates -the phosphorylgroup on the sensor kinase is then transferred to a response regulator --the phosphorylated response regulator canbind to DNA and affect transcription -inthe figure, the phosphorylated response regulator is a repressor protein |
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Multicomponentphosphorelay transfer system |
regulates sporulation |
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Controlof Flagella Movement |
-theamount of methylation on MCP determines the cells response to attractants andrepellants -bindingof methylated CheY tothe flagellarmotor results in clockwise rotation and tumbling of the cell -CheZ removes the phosphate from CheY sothat it release from the motor and allows the motor to rotate counterclockwiseand the cell to run |
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Translation |
-protein synthesis -mRNA is translated into protein -Key players: mRNA, tRNAs and ribosomes made of rRNAand proteins |
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Codon |
mRNAsequence of three nucleotides |
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Start codon |
•AUG •sequence at which translation starts •Bacteria – N-formylmethionine (f-Met) •Archaea & Eukarya – Methionine (Met) |
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stop codon |
•UAA, UAG, UGA •sequence at which translation stops |
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Openreading frame |
from start codon to stop codon |
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Genetic code |
•expressed in terms of mRNA codons • many codons can code for a single aminoacid |
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Rare exceptions to the universal genetic code |
-UGA codes for selenocysteine if there is a recognition sequence downstream of the mRNA that forms a stem-loop capable of binding SelB protein -similarly, UAG can code for pyrrolysine. |
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Aminoacyl Transfer RNA (tRNA) |
1. is attached to the amino acid corresponding to the codon 2. hasanticodon sequence that temporarily base pairs with mRNA codon duringtranslation |
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ribosome |
-brings together mRNA and aminoacyl tRNAs forprotein synthesis -goesfrom start codon to stop codon -movesalong the mRNA one codon at a time |
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Polysome |
several ribosomes can translate a single mRNA molecule simultaneously |
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Typesof Mutations |
•Induced –Physical ex. Ionizing radiation –Chemical ex. intercalating agents •Spontaneous –Errorsby DNA polymerase in DNA replication |
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Ionizing radiation |
–Penetratestissues, causes formation of ions that can break covalent bonds –Lowlevels create point mutations –Highlevels create large chromosomal mutations–Radiationdoses are cumulative –Examples: X-rays, cosmic rays, and radon 20,000+deaths per year from radon-induced lung cancer |
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IntercalatingAgents |
–Ex. Ethidium bromide –Insert between bases in one or bothstrands causing the helix to relax –If the relaxed strand is the template for replication, then an extra base will be inserted in the complementary strand -Ifthe intercalating agent is removed prior to replication then there will be noerror introduced upon replication -theintercalating agent creates a space between nucleotides -if the intercalating agent is introducedprior to replication into the template strand, then an insertion of onenucleotide will occur in the new strand opposite the intercalating agent -if the intercalating agent is introduced during replicationinto the new strand, this will prevent a complementary nucleotide from beingadded in the new strand opposite the template strand -when the intercalatingagent is removed there will now be a deletion. |
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Ethidium bromide (EtBr) |
-commonly used to stain DNA in gel electrophoresis experiments -when bound to DNA, EtBr fluoresces orange under UV light -EtBr stained DNA can easily be seen in a gel when you shine a UV light onto the gel |
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Acridines |
-a class of heterocyclic organic compounds used as fluorescent dyes (acridine orange), anti-tumor and anti-malarial agents (quinacrine) |
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TheAmes Test |
-the Ames test uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism -it is a biological assay to assess the mutagenic potential of chemical compounds |
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Ames test procedure |
-Cellsof the his- strain of Salmonellatyphimuriumhavea mutation that prevents the cells from making histidine (anamino acid) and therefore the cells require histidine inthe growth medium in order to grow- 1. A his- strain of Salmonellatyphimurium is grown in broth culture and mixed with S9extract (to mimic in vitro human conditions). This mixture is added to the surface of anagar plate with growth medium lacking histidine tocreate of lawn of bacteria covering the plate. 2. The chemical you want to test is added to apiece of filter paper (usually shaped as a disk). The filter disk impregnated with the chemicalis then placed on the lawn of bacteria. 3. The plate is incubated overnight. Because of normal spontaneous mutation,several his- mutants will revert to wild-type and grow up as colonies on the plate(1/500,000 cells). These colonies arecalled revertants. However, if the chemical on the disk is amutagen, there will be significantly more colonies (revertants)around the disk indicating the increase mutation rate in the bacteria near thechemical because of the chemical. |
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Point Mutations |
•A change in one or a few base pairs 1.Base-pair substitutions -Cause missense, nonsense, or silentmutations2.Base-pairinsertions/deletions -Cause frameshift mutations •Can occur anywhere in the genome •Can affect gene expression -2classes based on how the mutation affects phenotype 1.Forward mutation 2.Reverse mutation |
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Point Mutation Affect on Phenotype |
•Forward mutation –Changes a normal (wild-type = WT) gene to a mutant gene •Reverse mutation (reversion, backmutation) –Changes a mutant gene back to a completely WT or nearly WT gene |
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Effectsof Base-Pair Substitutions |
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Effectsof Insertions or Deletions |
Frameshift mutation – a shift in the open reading frame caused by insertions or deletions. |
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MutationRates |
•Replication error rates –Humans10e-5 –Bacteria10e-6 –DNAviruses 10e-4 –RNAviruses 10e-3 |
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Mechanismof SOS response |
1. DNA damage activates RecAwhich activates LexAprotease activity 2. The repressor LexAis self-cleaved, degrading it allows for transcription of many genes includingseveral DNA polymerases (II, IV, and V) needed to repair the damage 3. When damage is repaired, RecAis inactivated and newly made LexArepresses DNA repair genes. For example,uvrAis used in light-independent nucleotide excision repair to remove thyminedimers. |
