Biochem Test Ii

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Operon

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Genes whose activity is coordinately regulated

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Sigma 70-pol

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RNA Polymerase in prokaryotes, cannot bind if oppressor is present, requires cap to be added afterward to prevent from falling off

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Sigma subunit of RNA polymerase

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Recognizes promotor and intiates synthesis, lost after 10 BP

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Alpha subunit of RNA polymerase

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Binds regulatory sequences

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Beta Subunit of RNA polymerase

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-forms phosphodiester binds and binds DNA template

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RNA polymerase

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Has helicase activity

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Promoter sequence and TATA box

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High homology, allows RNA polymerase to bind to the DNA strand, mutation in the box prevents binding, but mutation downstream only slows RNA polymerase

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CIS-acting elements

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DNA sequence that controls genes on the same chromosome

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Trans-Acting

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DNA sequence that encodes for diffusible proteins that control genes on the same or different chromosomes

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Aminita Phallodes

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Death Cap, inhibits RNA Pol II

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Transcription Factors

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HAve DNA binding and Activation Domains, DNA binding section binds to DNA And activator region brings in the necessary proteins, seperated by a flexible protein

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TBP

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TATA Binding Protein, binds to TATA box and bends DNA into a more favorable conformation for binding

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TFIIH

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Has helicase functionality and also repairs mistakes in the DNA sequence

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TFIIF

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Recruits RNA Pol II to join near the TATA box

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RPD3 (and Sin3)

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Deacetylation of the DNA, Ume 6 is the repressor sequence

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Gen 5

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Acetylation of DNA Gcn 4 acts as activator sequence

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Transcription Mediator

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bends or twists chromatin so that initiation complexes have better access to the DNA sequence

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Activated Transcription Initiation COmplex

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Mediator, Transcription Activation Factor(acetylator), TBP, TFIIH, Pol II, TFIIF

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Competitive Binding ofwith activator (Repressor Mechanism)

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Repressor and activator share same bonding site

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Interaaction with activation domain of bound activator (Repressor Mechanism)

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Active sites interact preventing interaction with other proteins

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Interaction with General transcription factors (Repressor Binding)

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Repressor binds with another factor near TATA box

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Hormone Dependent Gene Activation

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Hormone binds to ligand binding site of transcription factor removing the inhibitor, moves intor nucleus and DNA binding site binds to DNA

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Model for Phosphorylation of STAT

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IFN-gamma binds to receptor causing JAK to dimerize, leading to phosphorylation of STATalpha, Phosphorylated pieces dimerize and are able to move in through nuclear pore and bind to the response element on DNA activating transcription

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Activation of NF-kB responsive genes

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TNFalpha and IL1 receptors activate TAK1 which in association with an unknown mechanism activates I-kB kinase, this then sequesters the NF-kB and this then binds E3 ligase in a phosphorylated form, the E3 is removed and the I-kB is removed by ubiquitin and proteosome, NF-kB is then freed and allowed to move into the nucleus initiating transcripition of genes (specifically the alpha subunit of I-kB)

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Southern Hybridzation

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DNA is cut using restriction enzymes and then run on a gel, stained and viewed, ofetn used in forensics, can detect thalasemias

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Northern Blotting

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Same as southern only RNA is used

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Dideocy Sequencing of DNA

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labeled nucleotides are added that stop replication at certain sites yielding fragments of varyling length with known ending points, allows for sequencing of DNA

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PCR-polymerase chain reaction

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DNA is dnatured and primers are added, repeated many times to duplicate DNA sequence

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DNA Mircro-array

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Genome wide expressions, cells are grown in two different conditions and a fluorescent dye is added to protiens, then placed in known sequence wells and depending on color shows presence or lack of protein in the two different conditions

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Dendogram

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Shows relationships between genes, smaller bar lengths mean closer relation

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Single Nucleotide Polymorphisms- SNP's

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small genetic variation in sequence, most found outside coding sequences, in coding sequence may lead to improper proteins,

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Restriction Length Polymorphisms

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Uses SNP's and restriction enzymes to cut at different places shows prescences of different alleles

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RFLP

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test using Restriction Length Polymorphisms to construct a gel pattern

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Microarrays

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PCR duplicates, and are added to chip, oligonucleotide arrays are either hybridized with labled targets that ate yhen analysed or hybridized with an unlabeld target and then single base extensions occur using didoxy nucleotides.

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Gene Therapy-Germ line

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Random insertion causes mutaion, wild-type can segregate from mutant

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Gene Therepy- Somatic therapy

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Provide gene in some somatic cells which restores some function, retrovirus(current)inserts DNA into the chromsosome randomly, may lead to problems, or Adeno virus (testing) that inserts extrachromasomaly eliminating insertional mutagenesis

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Adult STem cells

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Some may be induced to dorm tissues related to them but not the same as

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N protein (or Cro)

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Antitermination factor that binds to B-box loop preventing termination by bringing in additional proteins

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Rho

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Terminates sequence in absence of N protein

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Transcription termination in Eukaryotes

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RNA Pol I-polymerase specifice termination factor
Rna Pol III-terminates after polymerizing poly U
RNA Pol II- terminates at multiple sites beyond poly Asite, coupled to cleavage and polyadenylation

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Tat

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HIV protein with similar functionality to N protein

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Eukaryotic mRNA processing

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1. Transcription-capping at 5'
2. Cleavage at Poly A site
3. Polyadenylation at 3'
4. RNA Splicing

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mRNA capping

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Triphosphate at 5', cleavage of one phosphate occurs, GTP then binds and transfers on the Guanin and ond phosphate, methylines are added by Guanine 7 methyl transferase, and methyl group is added is added to first N

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Splice Sites

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Splice sites have high homolgy especialy in GU and Ag sequences telling spliceosomes where to cut

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snRNP's

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associate together to form spliceosomes, catalyze two transesterification rxn's cutting out introns and splicing exons, 1 spliceosome can go to multiple splice sites

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Mutations in Splice Sites

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Mutaion in splice sites may result in a mutation if splice sites change either including introns or cutting out exons

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Cleavage and Polyadenylation Model

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CPSP binds to poly a signal, CStF binds to downstream GU sequences with bound CPSP, PAP stimulates cleavage at poly A site, releasing cleavage factors and downstream RNA, PAP adds 12 residues, PABII accelerates rate of addition by PAP

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Nuclear export of mRNA

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Has protein factors that fall off inside and outside nucleus, and require proteins to aid in crossing back to origins

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HIV Rev protein

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Produced by splice HIV RNA allows unspliced forms of HIV mRNA to cross into cytoplasm, prevents degradataion

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miRNA

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micro RNA, regulate translation efficiency, can block translation, expression can be regulated

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siRNA

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short interferon RNA, manufactured, induce degradation of mRNA eliminating protein function, could be used in deisease treatment by targeting certain disease mRNA

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Stability of mRNA regulated by proteins

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Iron Responsive Elements-IRE- bind IRE-BP in low iron conditions preventing the mRNA from degrading and allows production of transferon receptor to bring Fe into cell.
Also can block translation of Ferritin mRNA that binds FE in the cella allowing it to be used.
Hemeglobin production is similar as Heme must be present for translation

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rRNA splicing

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2 methods, both are self splicing transesterification rxn's, group 2 is similar to spliceosome function, RNA has catalytic ability

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tRNA processing

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Introns removed
some sequences cleaved off
CCA is added to end
some bases are modified

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SRP receptor and Ribonuclear protein complex

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Binds ribosome to ER when signal sequence is present

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Translocon

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Membrane Protein that opens allowing protein to move into lumen of ER or other lumen

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Signal Peptidase

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Cleaves signal sequence from protein

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STA, SAIII

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Stop transfer sequences leading to NH3 in lumen, seperate signal sequence

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SAII

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Signal anchor leading to NH3 in cytosol, no seperate signal sequence

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SAII,STA,SAII,SAT
SAIII, SAII, STA, SAII

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cytosol, lumen, cytosol, lumen
Lumen, cytosol, lumen, cytosol

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N-linked Olihosacharides in ER

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Sugar added to Asn amino acid in protein and Glucose is either added or removed depending on protein folding, finished it is removed

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IRE1

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As monomers bind BiP chaperone protein, as dimers splice Hac 1 gene to make more BiP

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BiP

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Chaperone protein in ER lumen

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Mitochondria Proteins

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Fully synthesized proteins are recognized by import receptors, bring in translocons (TIM and TOM, proteins are refolded as they pass through membranes

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Peroxisomal Proteins

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Pex 5 binds protein and docks to Pex 14, Pex 10,12,and 2 allow protein to pass into lumen of peroisome

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Zellweger syndrome,

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defect in peroxisomal protein transfer, impairment of organs and early death

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Importin

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Moves protiens into nucleus, binds and moves in with GDP, GDP forms GTP and binds to importin after release and moves back out of nucleus

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Eportin

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Moves protein out of nucleus, Binds to GTP and protein and moves out into cytoplasm, binds to an importin and moves back into nucleus with GDP

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Golgi Maturation

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Cis, medial, trans

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ABO blood types

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Caused by different olligosacharide components, A-GalNAc, B-GAl, O-

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COPII

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Acts in vesicular transport btwn ER and Golgi, coats vessicles moving toward Golgi, falls off as nears golgi to allows SNAREs to attatch

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COPI

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Acts in vesicular transport btwn ER and Golgi, coats vessicles moving toward ER, falls off as nears ER so SNAREs can attatch

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pH gradient

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pH gradient effects the binding

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KDEL Receptor

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Has low binding affinity in the ER but high affinity in the Golgi lower pH, so that proteins that are sorted incorectly are returned to teh ER

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Lysosomal Targeting of M6P

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M6P receptors in Golgi and on cell surface, bind M6P and form a clathrin coated vessicle, uncoats and fuses with endosome (low pH) and releases, receptor then returns to either the cell membrane or the Golgi

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Tay Sachs

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defects phosphotransferase in formation of M6P residues on lysosomal enzymes in cis-golgi, leads to accumulation of glycolipids and other components leading to deformities, retardation, and early death

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Receptor mediated endocytosis: LDL

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LDL binds to receptor on cell surface, forms a clathrin coated vessicles, uncoats and fuses with endosome, releases at this low pH, receptor returns to surface

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Receptor mediated endocytosis: Trensferin Ccle

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Treansferin bound to Fe binds to receptor and forms a clathrin coated vessicle, uncoats and Fe is relased as vessicle fuses with low pH endosome, receptor with transferin returns to surface and releases transferin at neutral pH

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Actin Cross linkage

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cross linkage of actin leads to multiple functionalities within th ecell, providing many key roles

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Platelts and actin

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Actin monomers are bound by profillin, Ca+2 influx activates gelsolin, cleaves profillin, activated actin no forms filaments that spread and attatch to blood clot

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Dystrophin

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Binds to actin to form a structural role, actin provides some rigidity in muscle

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Critical concentration Cc

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Concentration at which actin or microtubules go from monomer to polymer form, causes treadmilling characteristics

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+ end

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Much lower Cc aids in polymerization

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- end

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much higher Cc leads to easier dissasembly

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Cytochalasin D

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Depolymerizes actin filaments by binding to + end where it blocks further addition of subunits

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Latrunculin

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Binds G actin and inhibits it from adding to the filament end

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Phaloiden

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Death cap mushreoom binds interface btwn f actin subunits locking together, stabilizes

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Cofilin

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Dissociates actin from the - end

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Profillin

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stimulates exchange from ADP to ATP to aid in addition of actin to + end

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Actin Polymerizatin at Leading edge

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Polymerization occurs here and results in movement of cell front, stained area remains the same as actin is added not actually moved

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Microrganisms that utilise actin rockets

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Listeria, Shigella, Rickettsiae, Caccina Virus

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Centrosome

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paired centrioles at 90 degrees

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Tubulin Dimer Structure

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Alpha tubulin contains permanently bound GTP, Beta tubulin contains GDP that is exchangable and hydrolizable with GTP, also has Taxol binding site

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Gamm-tubulin Ring Complex (gamma-TURC)

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Nucleation center near nucleus, often asscoiated with - end, caps it so that it is stablized

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Temperature and Microtubules

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Dcrease temp causes microtubules to dissasemble, return with increased temp-dynamic instability

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Catastrophe

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Stopping point of microtubule growth, highest point.

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Rescue

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Stopping point of microtubule dissassociation, lowest point

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Map2 and Tau

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MT stabilizers, often found in axons of nerves and other areas of important vessivular transfer, prevents dissociation of MT, Tau is often associated in high levels with Alzheimer's

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MT destabilizers

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Kinesin-13 and Stathmin, kinesins induce conformational changes and stathmin induces hydrolyses

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Taxanes

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Taxol-stabilize MT, treatment of Breast, ovarian, lung cancers and Kaposi's sarcoma
Block Cell Division

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Microrganisms that utilise actin rockets

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Listeria, Shigella, Rickettsiae, Caccina Virus

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Centrosome

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paired centrioles at 90 degrees

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Tubulin Dimer Structure

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Alpha tubulin contains permanently bound GTP, Beta tubulin contains GDP that is exchangable and hydrolizable with GTP, also has Taxol binding site

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Gamm-tubulin Ring Complex (gamma-TURC)

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Nucleation center near nucleus, often asscoiated with - end, caps it so that it is stablized

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Temperature and Microtubules

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Dcrease temp causes microtubules to dissasemble, return with increased temp-dynamic instability

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Catastrophe

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Stopping point of microtubule growth, highest point.

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Rescue

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Stopping point of microtubule dissassociation, lowest point

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Map2 and Tau

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MT stabilizers, often found in axons of nerves and other areas of important vessivular transfer, prevents dissociation of MT, Tau is often associated in high levels with Alzheimer's

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MT destabilizers

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Kinesin-13 and Stathmin, kinesins induce conformational changes and stathmin induces hydrolyses

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Taxanes

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Taxol-stabilize MT, treatment of Breast, ovarian, lung cancers and Kaposi's sarcoma

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Vinka Alkaloids

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Destabilize MT-
Vinblastine-lymphoma, testicular, Kaposi's Sarcoma
Vincritsine- Lymphoma, Leukemia
Vindesine-lung
block cell division

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Kinesin Structure

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Tail, Light Chain, Stalk, And Head
Head contains the MT binding sites and ATP/ADP binding sites

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Kinesin 1,2

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1 is normal kinesin, 2 is similar but with a heterodimer head, used in organelle transport

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Kinesin 5

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Bipolar, used in sliding microubules

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Kinesin 13

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Causes end dissassembly

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Kinesin Movement along MT

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first motor head binds B-tubulin with ADP attatched, Releases ADP and binds ATP, Binding of ATP causes conformational change leading to second head to move forward and bind releasing ADP, Original head then hydrolyzes ATP releasing Pi

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Axoneme movement

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9 doublet rings connected by nexin, Dyenins attatch to the MT pairs with an inner and outer arm, as dynein arms walk they cause the MTs to bend creating movement in the flagella/cilia, here MTs are a structural component

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Vessicle Transport in the Cilia

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Important in Primary cilia, kinesins transport vessicles dwon the cilia, critical for function, mistakes can lead to polycystic kindey diseas, diabetes...

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Mitosis and MT

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Microtubules dissociate during mitosis and return afterward, consist of polar, astral, and kinetochore microtubules, that help seperat daughter an dparent cells

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MT attatchment to the spindle

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Multiple MT attatch to the Kinetochore add in stability of connection, chromosomes seperated by dissociation near the MTOC, near the chromosome, and through dyneins that walk along polar microtubules, in addition the spindles are pulled farther apart by the astral MT pulling apart

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MPF

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Mitosis Promoting Factor, oscilates during the cell cycle, synthesized and degraded during each cycle, In mitosis, it is synthesized in prophase, MPF is in highest activity during metaphase, and lowest during telophase, regulated by APC/c which polyubiquitizes MPF during late anaphase,

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MPF phosphorylation

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Mitotic cyclin binds with CDK, Wee 1 adds one phosphate to Y, CAK adds one phosphate to T, Cdc 25 removes phosphate form Y and activates MPF

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MPF and Nuclear Lamina

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MPF phosphorylates the lamins in the nuclear lamina to break down the structure, Lamina returns through vessicles and create karyomeres, nuclei around the nucleus that aid in reformation

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APC in cell cycle

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Acts to break down anaphase in hibitor and cyclin B to exit the cell cycle

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Various cyclin CDK combinations throughout cell cycle

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Mid G1-Cyclin D/CDK 4 or 6
Late G1-Cyclin E/CDK 2
S-phase-Cyclin A/CDK2
Mitotic-Cyclin A or B/CDK1

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Passage of Restriction Point

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Cyclin D/CDK 4 or 6 begins phosphorylation of RB, this releases E2F to activate Cyclin E/CDK2 in late G1, Cyclin E/CDK2 then continues to phosphorylate RB and continue the cycle

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E2F and RB

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E2F acts to activate cyclin E/CDK2 to pass the restriction point of the G phase, RB prevents this, but when phosphorylated E2F becomes active

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Cyclin A/CDK2 and DNA synthesis

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Required for DNA syntheis, prevents pre-replication complexes from forming at multiple points, phosphorylates preinitiation complexallowing for continuation fo replication

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Checkpoint COntrols of Cell Cycle

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Intra-S Phase checkpoint-ensures DNA replication is complete b4 entering M-phase
Spindle Assembly Checkpoint-Ensures all chromososme kinetochores are attatched to spindle MT b4 anaphase
Spindle-position Cehckpoint-ensures all chromososomes are properly segregated to daughet cells b4 telophase and cytokinesis
DNA-damage checkpoint- detects damage to DNA throughout the cell cycle