molecular2 Flashcards
Initiation codon AUG interacts with tRNA.
in eukaryotes: methionyl trna, in bacteria: n-fmeth trna
Position of AUG at start: ____ at middle:_____
initiator, reg methionine
Shine Dalgarno is ____ of AUG, and functions to ___
upstream, attract ribosomes. it is 5’ UTR
Eukaryotes dont have a ribosome binding site (T/F)
True, so they have special cap on 5’ end of mRNA
Initiation includes
mRNA, tRNA, Ribosome
Elongation starts
once 2nd codon is used
5’ UTR
untranslated region before the initiator. These can hybridize with 16s
3’ UTR
downstream of the stop codon
P site
after initiaiton, aminoacyl tRNA binds to this site on the ribosome
Elongation adds __ one at a time to initiating amino acid
AA’s
First elongation step
binding second aminoacyl tRNA to the A site. This requires: EF-Tu, and energy from GTP
E site
exit, where tRNA exits ribosome.
Translation occurs through ____
elongation factor G
Peptidyl transferase
makes peptide bond with carboxyl and amino
Stop codons are:
UAG, UAA, UGA. these do not code for amino acids.
Release factors
recognize stop codons, and stop translation, releases polypeptide chain.
Peptidyl transferase cuts ____ when theres ____
polypeptide chain, stop codons
In between initiation and termination codons, is the
open reading frame
Transcription ___ primer dependent, replication ____ primer dependent.
isnt, is
Semiconservative replication
produces new DNA w each daughter double helix having 1 parental strand and 1 new strand
Without complete replication there is no ______
cell division.
Conservative replication
2 parental strands stay together, along with 2 new strands.
Dispersive replication
DNA is fragmented with both
Mutations can only occur during ______
replication, if DNA polymerase leaves a mistake behind
Mutation
silent mutation- occurs in 3rd base of codon, but makes same AA. Often changes are conservative like this, doesnt change the AA.
Sickle cell occurs when
Single base change of B-globin (hemoglobin). This causes substitution of wrong AA, distorting RBCs in low O2.
A change in a gene can change the protein product of a gene (T/F)
True
Sickle cell codon changes:
GAG (glutamate) changed to GUG (valine)
Molecular Cloning
combining a foreign gene in a plasmid. combines 2 DNA’s and makes it self replicating, which allows large production of target gene. Also, links eukaryotic genes to small bacterial/phage DNA, and inserts recomb. molec into bacterial hosts.
In between Nitrogenous bases are ______
covalent phosphodiester bonds.
Restriction endonucleases
-enzymes. usually a dimer
-made gene cloning possible
-discovered in E coli
-cut at sites within foreign DNA instead of chewing from ends, specifically cutting at the sugar phosphate backbones (at recognition sites)
-ligase recombines the cut ends by finding OH groups.
The frequency of cuts from restriction endonuclease is ____ frequent as recognition sequences are longer
less
Restriction modification
prevents restriction endonuclease from cutting host DNA. The mod is paired with methylases, they recognize and methylate the same sites. Methylation protects DNA
Boyer and Cohen experiment using restriction endonuclease
used EcoR1 to cut 2 plasmids, converting them to linear with the same sticky ends. DNA ligase joined the two pieces with covalent bonds. The enzymes, ligase, foreign DNA, and DNA being used, transform to desired bacteria.
Vectors
DNA carriers, allowing replication of recomb DNA. Carries foreign DNA to bacteria. Usually with 1 vector+ 1 piece of foreign DNA. Vector does not have origin of replication. Vectors can be plasmids or phages.
Multiple Cloning site
cloning sites clustered together. allows one to cut the vector and foreign gene with 2 different restriction enzymes. Uses directional cloning to know orientation for insertion.
Antibiotic resistance genes
allow for selection of bacteria that have a copy of the vector.
LacZ must have
plasmid transformation
Yeast artifical chromosomes and bacterial artificial chromosomes are used for
cloning huge amounts of DNA
Eukaryotic vectors
vectors designed for cloning genes to euk cells, or Ti-plasmid vector to carry genes to plant cells
Identifying clones with Probes
probes identify desired clone. The two types are polynucleotides and antibodies.
Factors supporting strand seperation
high temp, high organic solvent, low salt.
cDNA
complementary DNA or copy DNA that is a DNA copy of RNA. cDNA library is a set of clones with as many as possible of mRNAs in that cell type at a given time.
For successful cloning…..
synthesis of cDNA from mRNA template using reverse transcriptase- RNA dependent DNA polymerase.
Nick translation
removes DNA ahead of nick, synthesizes DNA behind nick. Result is moving the nick in 5’ to 3’ direction. The enzyyme used is E coli DNA polymerase 1.
cDNA: sticky ends
cDNA doesnt have sticky ends cleaved w restriction enzymes. Sticky ends will be made using terminal deoxy nucleotidyl transferase with one dNTP.
PCR
used to amplify DNA, creates a dna fragment for cloning. created by dr Kary Mullis. no need to add fresh DNA polymerase after each replication. Taq polymerase is used for PCR because its an enzyme that can be used at high temps.
PCR temps
boiling 99 deg to seperate ds DNA, 52-64 deg for primers/annealing, 72 deg for DNA polymerase to synthesize.
RT PCR
quantifies amplification of DNA as it occurs. As the strands separate, they anneal to forward and reverse primers, and to reporter probe.
Reporter probe
the 3rd primer. fluorescent tagged oligonucleotide that is complementary to part of a DNA strand, flores tag at 5’ end, flores quench at 3’ end. As PCR goes on from the forward primer, the 5’ tag is separated from the 3’ tag, allowing 5’ tag to fluoresce. can be quantitated.
Flourescence in RT PCR increases with _____
incorporation into DNA product.
Quencher of RT PCR
keeps surroundings dormant, stops from fluorescing. DNA polymerase separates the florescent from the quencher, allowing the F to fluoresce.
Quencher of RT PCR
keeps surroundings dormant, stops from fluorescing. DNA polymerase separates the florescent from the quencher, allowing the F to fluoresce.
Expression vectors
express genes. Used to put a foreign DNA into bacterium to replicate. These can make protein products of the cloned genes.
Bacterial expression vectors
Two required elements: a strong promoter (host RNA polymerase must be able to recognize), and a ribosome binding site by the initiating codon.
Inducible expression vectors
allows to express a toxic gene on the host cell
lac promoter
inducible, stays off till stimulated by inducer IPTG. Repression can be leaky, expression will still occur. To stop leak, use plasmid carrying its own lac1 repressor, this will stop RNA polymerase from binding.
Most vectors express _____
fusion proteins. not natural product of gene.
Oligo histidine expression vector
encodes 6 His. high affinity for metal ions like nickel . purified by nickel affinity chromatography.
His-tag can be removed by _____
enterokinase- which cuts histidine off desired protein. Result is eluted.
Bacterial expression Cons
-eukaryotic proteins in bacteria may be seen as foreign and destroyed.
-post -translational mods are different in bacteria.
-bacteria environment may not fold protein correctly.
-cloned euk proteins could be useless.
*Using yeast is safer
E coli can replicate both bacterial and euk cells (T/F)
True
Ti plasmid
used to transfer genes to plants. DNA stays in interstitial space.
-bacterial vector promoters and replication origins arent recognized by plant cells.
-plasmids with T DNA are used. (transfer DNA)
-Ti (tumor inducing) comes from bacteria that cause crown galls- plant tumors.
-crown galls occur if Ti is successful
Ti plasmid process
bacterium infects plant, transfers Ti plasmid to host, T DNA (may also contain gene of interest) integrates into plant DNA, T DNA synthesizes unusual organic acids (opines).
Encoded in tDNA:
Oxin, cytokinen, and opine
Gel electrophoresis
used to separate nucleic acids and proteins.
DNA gel electrophoresis
melted agarose is used with comb, electric current is run through gel at a neutral pH. DNA runs from neg to positive. DNA is negatively charged (because of phosophates). It will distribute DNA by their size, large to top, small to bottom, and stained with florescent dye.
Large DNA for gel electrophoresis is ____
fragile
Pulsed Field Gel Electrophoresis
used for DNA> 1kb. long pulses of current with short pulses in opp direction, allows to handle friction.
Protein Gel electrophoresis
Made vertically between 2 plates. polyacrylamide gel electro. performed with sodium dodecyl sulfate (SDS). small proteins move faster to anode. If mix is very complex, use also the isoelectric focusing gel- proteins electrophoresed through gel. Contains ampholytes for pH gradient. negative protein moves to isoelectric point. proteins are separated based on isoelectric points.
SDS
denatures protein, masks charge, provides negative charge
Ion exchange chromatography
used with pockets of charge. uses a resin to separate substances by charge. sample is loaded to column with resin.
Anion exchange chromatography
uses pos charged diethylaminoethyl groups (DEAE), that’ll mind to neg charged AA’s. Passes a soln of gradual ionic strength to column. uses salt gradient
Cation Exchange chromatography
negatively charged phosphocellulose resin attracts pos charged AA to seperat3e pos substances. Most proteins have net negative, but can still bond if they have significant pos charge
Gel filtration chromatography
protein size used as a basis of physical separation, uses porous whiffle ball type filtration. small molecules only will enter holes, so they will come out last because they follow path of holes, where as larger will go around and come out.
Affinity chromatography
enables purification of a biomolecule w respect to its biological function or individual chemical structure. can purify a certain molecule from a mixed sample. molecule will bind to a column resin coupled to affinity reagent. molecule of interest is retained.
