BICH 410: FINAL EXAM
69 Cards in this Set
| Front | Back |
|---|---|
|
Puromycin
|
aminoacyl-tRNA analog, binds at A site and acts as peptidyl acceptor, aborting peptide elongation
|
|
Tetracyclin
|
inhibits aminoacyl-tRNA binding at A site
|
|
Chloramphenicol
|
binds to 50S subunit, blocks the A site and inhibits peptidyl transferase activity
|
|
Streptomycin
|
binds 16S rRNA, interferes with binding of fMet tRNA to 30S
|
|
Cycloheximide
|
inhibits translocation of peptidyl-tRNA
|
|
Ricin
|
catalytic inactivation of 28S rRNA via N-glycosidase action on A4256
|
|
Diphtheria toxin
|
inactivates eEF-2 through ADP-ribosylation
|
|
construction of
cDNA library
|
1. mRNA produces cDNA through reverse transcriptase
2. we then cleave the cDNA and vector
3. we then ligate cDNA in the vector
4. then insert into E. Coli and the bacteria rapidly proliferates.
5. isolate DNA and cDNA library of active transcribed genes
|
|
identification and verification of
clone
|
Hybridization with radioactive probe
1. obtain agar with transformed bacteria
2. add nitrocellulose paper to agar plate and press to allow cells of each colony to stick
3. add alkali to disrupt the cells and expose the denatured DNA
4. incubate paper with a radioactive labelled probe
…
|
|
Topoisomerase
|
relieves positive supercoils
|
|
Helicase
|
helix destabilizing protein (causes strand separation)
|
|
SSB
|
stabilizes single strand regions
|
|
primase
|
makes RNA primer
|
|
DNA Polymerase III
|
synthesis of new strand
|
|
DNA polymerase I
|
removes primer
|
|
DNA ligase
|
seals nick
|
|
Reasons for high fidelity in replication
|
1. concentrations of dNTPs are balanced
2. polymerase reaction is 2 staged (base pairing--> polymerization)
3. 3'--5- exonuclease activity
4. repair enzymes
|
|
MutS and MutL
|
MutS recognizes base pair mismatch and forms complex with MutL. This brings MutH to the site and MutH binds to the hemi-methylated site. They all interact through DNA looping mechanism. MutH makes a cut on non-methylated strand. Exonuclease digests nonmethylated strand from cleavage site …
|
|
Methyltransferase
|
chemical mutagens react with bases and cause mutation. EX: Guanine reacts with alkylating agent and produces O6-methylguanine. Now guanine pairs with thymine rather than cytosine. Repair by methyltransferase.
|
|
Photolyase
|
UV radiation causes dimerization between thymine bases. Cyclobutyl ring is formed between C5 & C6 and normal base pairing is disrupted. Photolyase binds at dimer and uses energy of visible light to break cyclobutyl ring and restore function.
|
|
DNA glycosylase
|
A DNA repair enzyme that cleaves the bond linking a purine or pyrimidine to the deoxyribose of the backbone of a DNA molecule. (used in base and nucleotide excision repairs)
|
|
what is a gene?
|
specific unit of DNA that contains the info to specify synthesis of a single polypeptide or functional RNA (tRNA/microRNA)
|
|
RNA Polymerase I
|
transcription of rRNA
|
|
RNA polymerase II
|
transcription of rRNA
|
|
RNA polymerase III
|
transcription of tRNA and others such as 5S rRNA
|
|
Sanger Method
|
(dideoxy sequencing) DNA is divided into 4 separate reactions with same template, 4 dNTPs, DNA polymerase, and DNA primer. and a different ddNTP to each tube is added. They stop DNA polymerization at positions with that complementary base. Place fragments on lectrophoresis. Fragment sizes…
|
|
PCR reaction
|
used to selectively amplify DNA
primer, DNA, DNA polymerase and dntps are added.
DNA is heated and denatured at 94
annealing the primer at 54 to single stranded DNA
extension of DNA at 72, polymerase synthesizes DNA
|
|
DNA replication mechanism
|
1. Initiation at the origin and initiator proteins unzip the double strand
2. Elongation by polymerases. Each template for creating NEW strand. Primase adds RNA primers to the template strands, leading strand receives one primer, lagging receives several and is discontinuous and form oka…
|
|
mRNA splicing
|
removal of intervening sequences that interrupt the coding sequences of a gene and ligating of coding regions
|
|
mRNA splicing
|
-occurs in the spliceosomes via the llariat intermediate
|
|
alpha helix #'s
|
thickness: multiply by 1.5
turns: divide by 3.6
hydrogen bonds: subtract 4
alpha helix has hydrophobic AA's (valine, alanine, leucine, proline)
|
|
Beta form #'s
|
length: 3.4
BP per turn: 10.5
|
|
affinity chromatography
|
1. GST -tag affinity column. glutathione sepharose 4B. elute with glutathione to remove proteins
2. 6 x His tag affinity column. Ni-NTA sepharose. elute with imidazole.
|
|
experimental definitions of Vmax and Km
|
Vmax: maximum velocity when ES=Et. reaction velocity when all the enzyme is in ES conformation
Km: affinity of enzyme to substrate
|
|
Proteases
|
CNBr: Met (M)
Chymotrypsin: Phe, Trp, Tyr (F,W,Y)
Trypsin Arg, Lys (K,R)
Clostripan: Arg (R)
|
|
disaccharide linkages: lactose, maltose, sucrose
|
lactose: B (1-->4)
maltose: A (1-->4)
sucrose: A (1-->2) B
|
|
Starch and Cellulose linkages
|
starch: A(1-->4)
cellulose: B(1-->4)
|
|
F0F1 ATPase
|
complex located in the mitochondrial membrane. It uses a proton gradient to drive ATP synthesis.
F0 motor- ion translocation
F1 motor-ATP turnover
Protons bind to subunits of F0 domains and cause rotation, which powers synthesis of ATP by F1
domain.
|
|
nucleosome
|
a subunit of chromatin.
DNA wrapped around a histone core
this structure profoundly influences replication and transcription
|
|
Eukaryotic RNA Polymerases
|
I- transcribes rRNA
II- transcribes mRNA
III- transcribes tRNA and other RNAS such as 5S rRNA
|
|
Spliceosome
|
where mRNA splicing occurs; cuts introns out
|
|
Proteasome
|
degrades unneeded or damaged proteins by proteolysis
|
|
70S/80S ribosomes
|
protein synthesis in prokaryotes (70 S) and eukaryotes (80S)
|
|
Serine Mechanism
|
1. Aspartic Acid H-bonds with the Histidine making the nitrogen on the His much more electronegative
2. The electron pair on Histidine's N accepts the H from serine's -OH group and this coordinates the attack of the peptide bond
3. The serine -OH acks as a nucleophile and attacks the ca…
|
|
GroEL/ GroES
|
molecular chaperones that help fold proteins. unfolded proteins bind to hydrophobic binding patch on GroEL.
GroES is a cochaperonin protein that works in conjunction with GroEL.
|
|
Helicase
|
helix destabilizing protein and causes strand separation
|
|
Primase
|
makes RNA primer
|
|
3-->5 exonuclease activity of DNA Poly III
|
proofreading; excises the mismatched base
|
|
DNA ligase
|
seals nicks
|
|
topoisomerase
|
relieves positive supercoils
|
|
5-->3 exonuclease of Poly I
|
removes primer
|
|
How do leucine zippers interact with DNA?
|
the positive charged residues (Lysine or Arginine) in the DNA-binding region interact with the negative charges phosphate in the DNA backbone
|
|
what is the unique function of leucine zippers?
|
this region facilitates the dimerization of proteins by forming the hydrophobic sides of the helixes
|
|
What would happen if the leucine residues on the leucine zipper are mutated to serine?
|
the 2 helixes will not dimerize because the serine will have hydrogen bonds. this will NOT affect the DNA binding.
|
|
What will happen if Arginine residues are mutated to Glu?
|
Glu has a negative charge at 7 and WILL NOT bind DNA. (it will repel the phosphate backbone).
|
|
aminoacyl-tRNA synthetases
|
aminoacylation of tRNA
|
|
fMet-tRNA fmet
|
1. initiation of protein synthesis
2. has an anticodon that can bind with the start codon on mRNA
|
|
elongation factors: EF-tu, EF-G
|
EF-tu: helps aminoacyl-tRNA move onto free site on the ribosome
EF-G: catalyzes the movement of tRNA and mRNA through ribosomes
|
|
Initiation factors (1,2,3)
|
IF1- associates with 30S ribosomal subunit in the A site. Prevents aminoacyl-tRNA from entering
IF2-binds to initiator tRNA and controls the entry of that tRNA into the ribosome
IF3-required for 30S subunit to bind to the initiation site in mRNA
|
|
Release Factor 1 (RF1)
|
recognizes stop codons on the mRNA
|
|
Puromycin
|
Premature termination.
Mech: enters A-site by resembling an analog of aminoacyl-tRNA and truncates protein synthesis
|
|
Tetracyclin
|
binds 30S and inhibits binding of aminoacyl tRNA to mRNA-ribosome complex (blocks A-sites completely)
|
|
Chloramphenicol
|
prevents protein ELONGATION.
inhibits peptidyl transferase.
binds 23S rRNA of 50S preventing peptide bond formation.
|
|
Streptomycin
|
binds to 16S rRNA, interferes with binding of fMet tRNA to 30S
|
|
Cycloheximide
|
reduces translation by inhibiting peptidyl transferase
|
|
ricin
|
catalytic inactivation of 28S rRNA via N-glycosidase on the A site
|
|
Diphtheria toxin
|
inactivates EF-2 through ADP-ribosylation
|
|
how does IPTG induce expression of the protein?
|
1. IPTG binds to repressor protein
2. the binding causes a conformational change and releasing of repressor protein.
3. Transcription is reactivated after the repressor is released from the binding site near the promoter.
|
|
3 enzymes involved in ubiquitin proteasome pathway
|
E1: U-activating enzyme
E2: U-carrier protein
E3: U-protein ligase
|
Signal Transduction - Flashcards