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87 Cards in this Set
- Front
- Back
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Name the 3 genes in the lac operon.
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LacY-lactoase permease- creates the transporter for uptake of beta gal
LacZ-beta galactosidase- LacA-thiogalactoside transacetylase - acetylates nonmetabolic beta gal |
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What is it called where the repressor binds?
how many does lac have? and where are they |
the operator 2 -- 1. adjacent to the promoter 2. within the coding region of lacZ
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Since lac operon has 2 operators how many repressors does it need?
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1 - the repressor - encoded by lacI binds to both operators
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What happens when the repressor binds in lac operon?
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causes the DNA to form a stable loop which blocks initiation because it binds to both operators
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What does beta galactosides act as?
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as inducers to cause the repressor to dissociate
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What is the inducer for lac operon?
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allolactose -- binds to lac repressor, causes conform change and it dissociates from operator
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When is the lac operon transcribed at max?
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When beta galactosides are the only carbon source
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What is method which glucose represses lac operon? how does it work? what is the activator?
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catabolite represssion- a weak promoter is promoted to a stronger one by an activator in the absence of glucose --- activated by CRP(cAMP regulatory protein) or CAP
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What is the signal for the activator to bind to the operator?
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the presence of cAMP -- cAMP binds to CRP which becomes the activator and bind to the operator
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How is cAMP produced?
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main C source w/o glucose - Enzyme III (a glucose transport enzyme) transfers a phosphate to adenylate cyclase and -CRP-cAMP increase
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What is the pathway for the production of cAMP and the CRP activator?
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low glucose -- activates EIII to phosphoralate andenylate cyclase -->cAMP cAMP then binds withe CRP to create the activator!
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What does CRP/cAMP do/fit?
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the alpha helix of each monomer of the cAMP/CRP dimer fit into the major groove of DNA
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When does the activator/repressor bind to the operator in lac operon?
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activator - when there is no glucose
repressor - when there is no lactose |
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What are 3 RNA processing --post transcriptional modification
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1. removal 2. additon of nuc
3. covalent modification of bases |
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tRNA processing-
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mature tRNA molecules are generated by processing the primary transcripts
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What cleaves the primary transcripts to mRNA?
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RNAases
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What are the 3 steps in tRNA processing.
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1. RNAaseP cleaves the primary sequence forming the 5' end of tRNA
2. RNAaseD cleaves the primary sequence froming the 3' end of the tRNA 3. tRNA nucleotidyl transferase - adds CCA to the 3' end |
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What does ribosomal RNA processing include?
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1. methylation and cleavage by endonucleases
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What is the length of prokaryotic rRNA primary transcript? -what does it includE?
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30S
includes 16S, 23S, 5S |
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Does pro do mRNA processing?
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No - because they happen at the same time --translation and transcription
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Why are euk mRNA processed?
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to add stuff the ends so its doesnt get degraded as fast
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What are the 2 mRNA processing that happens?
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1. 5' end cap (which is required for splicing and when the rib binds)
2. 3' poly A tail |
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How do you form the 5' cap?
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1. remove the 5' Phosphate 2. The phosphate created forms GTP which is added to the 5' end (cap)
3. N-7 base and others methylated |
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How do you create the poly A tail?
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poly A polymerase adds up to 250 A -- 1. RNA pol II synthesis a pol A signal 2. CPSF binds with RNA endonuclease, cleaves transcript and poly A pol binds
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How far to genes normally span? how long is mRNA
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3400 base pairs - mRNA wiht poly A =1050
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How long is the poly A tail?
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up to 250 A
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What is similar is all mRNA precursors?
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nucleotide sequence at splice sites
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What is the spliceosome made out of/
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45 proteins and 5 snRNA
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What are snRNPs made out of?
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protein and snRNA
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Genetic code - meaning of unambiguous and degnerate
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unambiguous - each codon only codes for 1 AA
degenerate - each aa can be coded for more than 1 codon |
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How many nucleotides are typically needed to code for an AA?
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usually 2 but 3 definately
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What are the stop codons?
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UGA, UAA UAG
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What is the structure of tRNA?
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4 arms and 1 acceptor stem --when aa attaches , 1 anticodon arm - contains the anticodon
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Where is the wobble position? What is its funciton?
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the 5' end of the anticodon -- allows a tRNA to recognizes more than 1 sequence -so you might not have to have 20 tRNAs
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isoacceptor tRNA molecules-
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needed because several condons call for the same aa
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aminoacyl -tRNA - What catalyzes this reaction? how many different ones do we have?
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tRNA with aa attached to the 3' end of the tRNA -- aminoacyl-tRNA synthetases =20 1 for each aa
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What enzyme puts together the activated tRNA?
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synthetase - binds to ATP and the AA and then finds the correct tRNA based on structure - may recognize anticodon and acceptor stem
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What is a special property of active tRNA synthetases -
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some can proofread --common error - valine instead of isoleucine
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What direction does the ribosome move? what direction is protein madE?
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5--3 along mRNA
N to C direction |
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Polysome--
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mRNA that is translated by more tha n 1 ribosome at a time
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What does the initaition complex in translation consist of/
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1. ribosome subunits (2)
2. mRNA template 3. tRNA - initiator 4. protein initiation factors |
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What amino acid has to have 2 tRNAs/
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met -- 1. for initiation 2. for internal met
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What is the names of the initiator for pro and euk?
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pro -N-formylmethionyl
euk -methionyl |
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What does the SD sequence bind to?
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the 30S ribosome and complementary base sequence at 3' end of 16S rRNA
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What can multiple SD sequences in one mRNAresult in?
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polycistronic -- mRNA carries more than one gene like operons
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What are required to form the initiation complex? how many does pro and euk have
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initiation factors
pro -3 euk - 8 or more --they catalyze assembly of protein synthesis complex at initiation condon |
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Where is the Shine-Dalgarno seq?
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upstream of initation and only in Pro
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What does euk IF 4 do?/ What is its name?
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Cap binding protien - binds to the 5'' cap of euk mRNA
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What proteins form the preinitiation complex? What does it do?
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40S rib, active tRNA, and other == searches mRNA 5-3 for initaitor codon (AUG)
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What signals the 60S to bind?
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when the met-tRNA binds to AUG
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What are the 3 steps of elongation?
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1.positioning of the correct aa-tRNA in A site
2. formation of peptide bond 3. translocation of mRNA codon by codon |
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What is bound to all aa-tRNA except initatior aa? -what do they do?
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Elongation factors -they help the correct aa get to the a site
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What all binds at the a site? when is it stabilized?
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ternary complex of EF-Tu-GTP-aa-tRNA -stabilized if the aa-tRNA matches the codon
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When is the GTP that is attached to EF-Tu hydrolysed? what does this do?
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When it pairs the correct aa-tRNA to the correct codon - this causes a conformational change in EF-Tu and it leaves the ribosome -FUELS THE TRANSLOCATION OF THE EPA SITES
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How is EF-Tu recycled?
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To recycled EF-Tu you need to replace the GDP with GTP and EF-Ts does this!
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peptidyl transferase-
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creates the peptide bonds in the aa that are in the A and P site in the 60S -- catalytic activity from 23S rRNA
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Where is the growing peptide chain leave the ribo?
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through a tunnel in the 50s
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How many pro releasing factors are they? what are they used for
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3 -- when a stop codon comes up and it doesnt call for a aa - a RF binds and causes hydrolysis of the peptidyl t-RNA to release the polypeptide chain
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How many high energy phosphate bonds are cleaved for each amino acid added? how many molecules are used?
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4 - But only 3 molecules ATP-AMP (2 Bonds) 2GTP- 2GDP
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T/F -- All proteins are translated at the same rate.,
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F - it depends on codon usage, sequence at initiation site, and secondary structure in mRNA
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What does each operon for ribosomal proteins also encode?
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a repressor that inhibits translocation of its operon
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What is ribosomal protein syntheis coupled to? how/
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ribosomal assembly --because ribosomal proteins that inhibit translation bind more strongly to the RNA and when that proteins concentration increase the translation will stop
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How is globin synthesis regulated?
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by translation initiation - requires stoichiometric amounts for syntheis to occur -- HCL phosphorlated EF2 when heme is low so globin synthesis cant occur - high heme interferes with HCL
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What are miRNAS used for?
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regulators of gene expression at the post transcriptional level. -regulate mRNA stability and translation
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What are miRNAS made from? where? How do they work?
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non or coding regions of DNA -processed in the nucleus and transported to cytoplasm- can produce hairpins
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What processes miRNAs and how?
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Dicer = one strand is degraded and other complexes with protein to form miRNA - some miRNAs promote translational repression or deadenylation of mRNA
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What is the pathway for a newly formed protein in euk?
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from cytoplasm to ER to golgi to final location
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Where are proteins that leave the cell created?
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in the rough ER luman
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How does a protein know to leave the cell?
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a signal peptide is on the n term and signals the protein to cross a membrane
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What is a common glycosyltion reaction/
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the covalent attachment of a complex oligosaccharide to the side chain of asp
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Recombinate DNA molecules-
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constructed with DNA from different sources
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What are the 3 types of cloning vectors/
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1. plasmids 2. bacteriophages 3. small artifical chromosomes
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Plasmid Vectors-
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for small inserts - replicate independently -transformation
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Bacteriophage Lamda Vectors-
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commonly used in bacteria - transfection is more efficient - disadvantage - first have to pack it into viruses
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Shuttle Vectors -
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Can be used to replicate in pro and euk -can transfer recombinate DNA between pro and euk
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Yeast Artificial Chromosomes
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Large DNA fragments - must be linear and contain euk origin, centromeres and telomeres
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Selection -
Screening-0 |
Selection -cells grown under conditions where only cells with insert will survive
Screening-transformed cells are tested for the presence of the recomb DNA |
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insertional inactivation-
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insertion of the DNA fragment within the coding region of a gene will inactivate that gene
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DNA library-
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consist of all recombinant DNA molecules generated by ligating the DNA fragments into vectors- contains both expressed and non expressed DNA
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What kind of restriction digestion do you want for forming DNA librarys? what the most common example in humans/
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Partial -dont want them to cut a lot - want to make sure each gene is included - BAC
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cDNA library=
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represents all the mRNA - use reverse transcriptase -- identification by the poly a tail on mRNA - depend on cell type
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What is the hardest part of cDNA?
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screening for the desired recombinant
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prokaryotic expression vectors-
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plasmids that have been engineered to contain regulatory sequences for transcription and translation
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RFPLs-
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genetic analysis based on variations in length of genomic restriction fragments
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How are RFPLs detected?
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by incubating fragmented DNA of many indivuals with a cloned DNA probe
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Site directed mutagensis of cloned DNA-
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allows you to change an AA -- can introduce a mutation to a gene
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How do you do mutagenesis?
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1. anneal primer with mutation to a strand with target gene
2. replicate using primer -want mutation incorporated in to a strand 3. transform in to cell or keep repeating using mutant primers |
