Cell Bio Test 2

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DNA: replication, transcription, translation

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Central Dogma: DNA -->replication--> transcription-->RNA-->translation-->protein
Griffith Experiment: 1. Injected pathogenic (S) and non-pathogenic strain (R) of S. pneumonia into mice 2. S strain: kills mouse, R strain: mouse lives 3. Heat killed S strain injected: mouse lives. 4. R strain w/ heat killed S strain injected: mouse dies 5. S strain from #4 infected: mouse dies Conclusion: S strain transformed R strain into pathogenic
Avery, McCarthy,& MacLeod: 1. S strain cells broken up and contents were separated (RNA, DNA, protein, lipid,carb) 2. cell components were put into test tubes w/ R strain cells Conclusion: R strain cells w/ S strain DNA was transformed into S strain
Hershey and Chase: Experiment: Is it protein or DNA that contains heritable material ? 1. Viral DNA is labeled w/ 32P and protein w/ 35S 2. Virus put w/ E.coli to infect 3. Viral heads sheared off bact. 4. Bact centrifuged and will settle at the bottom Conclusion: infected bact had 32P not 35S therefore heritable material is in DNA not protein
double helix: - ATCG nucleotides - sugar-phosphate backbone - bases held together by H-bond >A-T: 2 H-bond >G-C: 3 H-bond
3'-5' phosphodiester bond: 3' OH binds to 5' phosphate group
DNA structure: - developed by Watson and Crick - right handed twist - 10 bp per turn - major and minor grooves
DNA width: - 20 A diameter
Major grooves: Looking at the DNA straight on: wider portion of DNA - important for DNA binding proteins
Nucleosome: - DNA wrapped histones - 146 bases wrapped around histones - keeps DNA from getting "tangled"
Linker DNA: ~50 base pairs b/w nucleosomes - links get digested but not the DNA wrapped around histones b/c it's inaccessible
DNA Packing: Chromatin and Chromosome
Euchromatin: lightly packed chromatin
Heterochromatin: tightly packed chromatin and lightly expressed
Chromatin remodeling: loosens DNA to allow for gene expression - ATP-dependent DNA remodeling complex moved the DNA thru the nucleosome to make it available
DNA Replication: biological process that occurs in all living organisms and copies their DNA
Characteristics of DNA replication: - templated - primered - semi-conservative - bi-directional - origin of replication - built 5'-3' - initiation is tightly controlled
Meselson and Stahl: experiment to determine model of replication 1. bact grown in heavy N (15N) 2. bact transferred to normal N (14N) 3. DNA is isolated and centrifuged 4. heavy N-DNA will be closer to bottom & normal N-DNA closer to top. 5. normal N-DNA band got bigger and heavy N-DNA band remained the same size Conclusion: replication is semi-semiconservative
replication origin: - site where replication begins - composed of many A-T b/c of weaker H-bonds - bi-directional
Replication proteins: - helicase - primase - single stranded binding proteins - DNA polymerase - sliding clamp - topoisomerase - ligase - telomerase
Helicase: - motor protein - "walks" down DNA strand - breaks H-bonds - ATP dependent
Single stranded binding protein: prevents hairpinning of the lagging strand
Primase: - RNA polymerase (not from transcription) - uses DNA as template - makes primer 10 bp long - primer made 5'-3' - cannot proofread b/c primer will be taken out at the end of replication
Size of full twist in DNA: 34 A
Distance between base pair: 3.4 A
Minor groove: Looking at the DNA straight on: narrower portion of DNA
Chromatin: "beads-on- a string
Chromosome: - 30nm fiber (packed in groups of 6) - fiber gets folded as series of loops (extended form) - loop series are then compacted (condensed form)
DNA polymerase: - catalyzes phosphodiester bond - binds the 3'-OH to the 5' alpha phosphate - needs a free 3' end to begin adding nucleotides - releases pyrophosphate (PPi) + energy
Sliding clamp: - dimer - secures DNA polymerase to DNA strand
Topoisomerase: - relieves over-winding (mechanical strain) 1) cuts 1 strand ahead of fork 2) allows it to untwist 1 turn 3) reattaches strand - ATP independent
Okazaki fragments: - only on lagging strand - 1000-2000 base pair (pro) - 100-200 bp (eu)
lagging strand: - contains "nicks" from the Okazaki fragments - the nick are a result of a lack of a phosphodiester bond
Synthesis of the lagging strand: 1. primase attaches a primer 2. sliding clamp attaches to DNA strand and to DNA polymerase 3. polymerase adds nucleotides till it reaches the primer of the last strand 4. sliding clamp release polymerase 5. primase attaches more primers 6. sliding clamp attaches to the primers 7. DNA polymerase attaches to the sliding clamp and adds nucleotides
ligase: - ATP dependent - seals the 'nick' in the backbone
telomerase: - adds series of repeats at the end of template strand - allows the completion of DNA synthesis on the lagging strand - series do not code for anything - series: GGGGTTA
Removal of primers: - repair mechanism for intentional problems - nuclease: removes RNA primers - repair polymerase: replaces RNA w/ DNA - DNA ligase: makes the phosphodiester bond
Proofreading: - Done by DNA polymerase 5'-3' - fixes unintended problems from 1) phosphodiester bond is cleaved by a 3'-5' exonuclease 2) adds correct nucleotide 3) creates a new phosphodiester bond
DNA replication (w/o mismatch repair): Error rate: 1 per 10,000,000 nucleotides
DNA replication (w/ mismatch repair): Error rate: 1 per 1,000,000,000 nucleotides
Error rate of DNA polymerase: 1 in 10,000 base pairs
DNA Repair: mismatch and excision If no repair: - in sex cells: inherited disease - in regular cells: cancer
Mismatch repair: - errors from DNA replication - enzyme complex recognizes strand w/ mistake by the nick present on the new strand - cuts out new strand from the error the nick - DNA polymerase and ligase repair gap
Excision repair: - errors from the environment (ie: UV) - different set of enzymes to id different types - DNA polymerase and ligase involved in correction.
Types of DNA damage or errors: Depurination, deamination, thymine dimer, nonhomologous end joining
Depurination: removal of a purine base from DNA strand causing deletion
Deamination: removal of an amine group on C to yield U that could lead to an insertion of A instead of G
Thymine dimer: - caused by UV light - 2 adjacent T --> T--T - leads to deletion since polymerase will only add one A
Non homologous end joining: - accidental break in double strand - nuclease cuts out ends to make them even - ligase binds the strands again - net result: double-stranded break repaired w/ deletion of nucleotides at repair site
RNA polymerase: - binds to DNA at promoter - unwinds &read the DNA as it synthesizes RNA - transcribes 5'-3' - three types: Pol I-III
RNA Pol I: - synthesizes rRNA
RNA Pol II: - synthesizes: mRNA and snRNA (small nuclear)
RNA Pol III: - synthesizes: tRNA and snRNA (small nuclear)
mRNA: - carries genetic info to ribosome for protein syn - contains introns
rRNA: - scaffold for ribosome - catalytic role in translation - no introns
snRNA: - splicing - parts of mRNA introns
coding stand: strand that is not used for transcription
template strand: strand that is used for transcription
Transcription: initiation (pro): - sigma factor binds to promoter and tell pol II which direction to go - pol II binds at -35 box and the -10 box
Polycistronic: - one gene codes for multiple proteins - proteins are probably related.
Eukaryotic transcription: - Pol II cannot initiate transcription on its own - transcription factors (TF) are required
Transcription factors: - accessory proteins - assemble on promoter to help bind RNA Pol II - different for each polymerase
TATA Box: - found in minor groove - (-25) bp upstream from start site - must have promoters
TFIID: - distorts DNA to allow binding of TF and Pol II - TATA Box Binding Protein (TBP) binds first
TATA Box Binding Protein: - subunit of TFIID - binds to the TATA box & distorts DNA - single peptide chain w/ 2 very similar domains
Sequence of TF binding: 1. TFIID (TBF) 2. TFIIB 3. TFIIE 4 TFIIH 5. RNA Pol II w/ TFIIF & other TF
5' cap (Capping): - modified Guanine (7-methylguanosine) - attached backwards (5'-to-5' triphosphate bridge - happens in the nucleus - prevents nucleotide subtraction - recognition by the ribosome - identifier to export mRNA out of nucleus
Splicing: - occurs after capping and polyadenylation - removal of introns
Exons: - coding region - expressed as protein
Introns: - non coding - intervening sequence
sequences needed for intron removal: AGGUAAGU UAAC UAGG
snRNP: snRNA with proteins
Spliceosome: - larger RNA/protein hybrid that cuts out introns
Splicing mechanism: 1. U1 snRNP attaches to AGGUAAGU, U2 snRNPto UAAC 2. more snRNPs attach to form a lariat b/w an A and the cut 5'end of the intron 3. lariat is removed and degraded in the nucleus
What does splicing offer?: - allows for different versions of the protein by splicing all the introns and some exons depending on cell type
codon: - group of 3 nucleotides the code for an aa - degenerate: impossible to take a protein sequence & figure out the base sequence - redundant: many codons for one aa - universal
stop codons: UAA UAG UGA
Start codon: AUG (methionine)
Reading frame: - different possiblilties - one correct that codes for the right message
Open Reading Frame: correct reading frame
Transfer RNA: - 75 - 90 nucletides - links aa to codons - complicated 3D structure - more than one for ea aa - showed as a clover but actually more "L" shape
wobble: - one tRNA may recognize multiple codons of the same aa - tolerate mismatch on the 3rd position of codon -
Ribosome: - contains binding site for mRNA and three for tRNA
A site: - aminoacyl - entrance of tRNA w/ aa
P site: - peptidyl - tRNA holding the growing peptide chain - formation of peptide bond & breaking tRNA-aa bond
E site: - ejection - tRNA w/o aa and then leaves
Translation: initiation (eu): - ribosomes bind to 5'cap - moves down mRNA till it reaches AUG - mRNA codes for one gene
Translation process: initiation: 1. small ribosomal unit w/ inititaion factors and Met (initiator tRNA) move down mRNA for AUG 2. initiation factors dissociates & large ribosomal unit binds to small unit 3. tRNA binds to A site and tRNA w/ Met moves to P site 4. peptide bond is made
Translation process: elongation: 1. from step 4 of initiation, the tRNA that had aa moves to the E site and the tRNA with growing peptide chain moves to the P site. 2. another tRNA w/ aa that corresponds with the code on the mRNA. At the same time the tRNA in the E site gets released 3. peptide bond forms b/w aa of the P site tRNA and the aa of the tRNA in A site. 4. The tRNA w/o aa moves to the E site and the tRNA with growing peptide bond moves to P site. The process repeats till the ribosome reaches the stop codon
Translation process: termination: 1. release factor binds to stop codon in A site 2. release factor caused addition of OH to the last aa 3. polypeptide chain is released 4. ribosome dissociates and releases the tRNA and mRNA
Polyribosomes: - multiple ribosomes bound to a single mRNA - increases overall rate of protein production
Translation process in pro: - translation begins as soon as the leader sequence is transcribed - ribosomes translate before transcription is done
controllable genes: - expressed as needed - dependent on cell conditions
control of gene expression: - transcriptional - post-transcriptional
transcriptional: - transcriptional control - RNA processing control
post-transcriptional: - RNA transport & localization control - mRNA degradation control - translation control - protein activity control
Operon: - collection of related genes that are expressed & regulated together as a single mRNA - either all on or off ie: tryptophan and lac
Trp Operon: - makes enzymes for tryptophan production. - low trp turns on the gene by having an inactive repressor because trp binding to the repressor activates it. - high trp turns off gene by tryptophan binding with the repressor on the operator of w/in the promoter, preventing the RNA pol from binding.
purpose of lac Operon: - Bacteria prefer glucose because more efficient for glycolysis - Genes for glycolytic enymes are always expressed - when gluc is not present, bact sense presence of other sugars & express genes that can metabolize them
lac Operon: - expressed when lact is present and gluc is not - breaks down lactose into galactose and gluc to utilize gluc for metabolism
genes of lac operon: - beta-galactasidase - permease - transacetylase - repressor
beta galactasidase: - hydrolyzes lactose --> gluc + galactose - made form lacZ gene
permease: - membrane transport protein to pump lactose into cell - made from lacY
transacetylase: - enzyme that transfers an acetyl group to lactose - made from lacA
repressor: - bound to the promoter/operator region of lac operon when lact is not present (active) - lact causes repressor to dissociate from promoter/operator - tetramer - made by lacI
CAP: - catabolic activating protein - forms a complex with cAMP - bind to promoter to enhance transcription - low gluc = more cAMP
two theories for co-regulators: - stabalization of initiator complex - chromatin remodeling to make the gene more accessible.
chromosome structure in gene expression: - genes near telomere or in tightly packed DNA are not expressed well -HAT and chromatin remodeling complex can make DNA more or less accessible
HAT: - recruited by activators or repressors - specific histone modifications
Chromatin remodeling complex: - winds or unwinds DNA across histones
Epigenetics: histone modifications passed on from parental to daughter cells
DNA methylation: - methylation of cystine stops RNA Pol in transcrption - ensures genes are regulated
IRE: - Iron responsive element - site where IRE-BP binds
IRE-BP: - IRE binding protein - encourages transferrin mRNA - inhibits ferritin mRNA translation - falls off mRNA when Fe binds to it
Transferrin (Fe receptor) mRNA: - makes cell surface receptor that takes Fe into cell. - IRE is in poly A tail - low [Fe] --> IRE-BP bound to mRNA --> preserving the mRNA --> translating more protein to bring in more Fe - high [Fe] --> Fe binds to IRE-BP --> IRE-BP falls of IRE --> mRNA gets digested
Ferritin mRNA: - makes protein to store excess Fe - IRE is in 5'cap - low [Fe] --> IRE-BP bound to mRNA --> repressing translation - high [Fe] --> IRE-BP falls off mRNA --> initiating translation
Riboswitch: - short sequence of RNA that changes confirmation when bound to small molecules - low guanine --> 2nd struc mRNA --> promote transcription of gene for guanine biosyn - high guanine --> guanine binds to mRNA --> changing its confrimation --> stopping transcription
micro RNA (miRNA): - regulates stability & translation of mRNA - 40,000 in humans - regulate 1/3 of all human coding genes
micro RNA function: - made in nucleus - diced into smaller piece - assoc w/ RISC to guide it to mRNA with complementary bases - miRNA bind to complemetary bases and RISC cuts off the base pairing region - extensive match --> rapid mRNA degradation - less matching --> translation reduced and mRNA taken to other parts of cell to be further degraded.

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