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85 Cards in this Set

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DNA Polymerase Catalyzes
Catalyzes phosphodiester bond formation

- Catalyzes the step-by-step addition of deoxyribonucleotide units to a DNA chain
(DNA)n + dNTP <--->
(DNA)n+1 + PPi

(dNTP=deoxyribosenucleotide, PPi= pyrophospate ion)
Characteristics of DNA synthesis
1) The reaction requires all _ activated precursors: and _
The reaction requires all four activated precursors:

deoxyribonucleoside 5'-triphosphates:
dATP, dGTP, dCTP, TTP.
and Mg2+
Characteristics of DNA synthesis:
2) DNA polymerase requires a ___ to synthesize a product:
DNA polymerase is a ____ directed enzyme.
2) DNA polymerase requires a template to synthesize a product that is complementary to the template.

DNA polymerase is a template-directed enzyme.
DNA replication proceeds from _' to _'
DNA replication proceeds from 5' to 3'
DNA Replication:

DNA must be bound by a ____ having a free _'- __ group

This group nucleophiliclly attacks ____________ and forms a __________ and releases a ______.

This reaction is driven by ______.
DNA must be bound by a primer having a free 3'-OH group.

The free 3'-OH group on the primer nucleophiliclly attacks the innermost phosphorous on the nucleoside aand forms a phosphodiester bond and releases pyrophosphate.

the reaction is driven by hydrolysis of pyrophosphate
RNA polymerase that copies viral RNA from an RNA template
RNA-directed RNA polymerase copies viral RNA from an RNA template.
Genetic information in retroviruses flows from ____ to ____.
Genetic information in retroviruses flows from RNA to DNA.
Retroviruses contain _ strands of ___ which upon entering a cell it will infect is copied into ___ through the action of a viral enzyme called ________.
Retroviruses contain 2 copies of a single stranded RNA molecule. On entering the cell the RNA is copied into DNA through the action of a viral enzyme called reverse transcrpitase.
mRNA:
1) photocopy of genetic information
2)this genetic information is translated to synthesize functional proteins using different RNAs
Restriction Enzymes are:
Restriction Enzymes are precise, molecular scalpels that allow an investigator to manipulate DNA segments.
Restriction Enzymes recognize:
Restriction Enzymes recognize specific base sequences in a double helical DNA and cleave, at specific locations, both strands of that duplex.
Restriction Enzymes are ___ about their cleaving locations.
Restriction Enzymes are symmetrical about their cleaving locations
RFLP
RFLP (Restriction fragment length polymorphism)- can detect diseases
___ Blotting
can be used to follow the inheritance of certain DNA genes.
DNA sequencing: the precise ____ sequence can be determined and from that 3 things can be determined:
DNA sequencing: the precise nucleotide sequence can be determined and from that
-gene architecture
-control of gene expression
and
-protein structure can be estimated
PCR (______): amplifies
PCR(Polymerase chain reaction): amplifies specific DNA sequences
PCR is carried out by adding the following to a solution containing___
PCR is carried out by adding

-the target sequence

- a pair of primers which will hybridize with the flanking sequences of the target

-all four dNTP's

-a heat stable DNA polymerase
A PCR cycle contains the following steps:
1) Strand Separation: The two strands of the parent DNA are separated by heating to 95*C for 15 seconds

2) Hybridization of the primers: the solution is then abruptly cooled to 54*C to allow each primer to hybridize to a DNA strand. One primer hybridizes tothe 3' end of the target on one strand and the other primer hybridizes to the 3' end on the complementary target strand. (parent DNA duplexes do not occur because of the large excess of primesr)

3)DNA synthesis: The solution is then heated to 72*C, the optional tempurature for Taq DNA polymerase. This heat stable polymerase elongates both primers in the direction of the target sequence (5' to 3'). This takes place on both strands.

This is repeated again and again. Increases exponentially.
For PCR:
The sequence of the target needs/does not need to be known:

the target can/cannot be much larger than the primers

primers do/do not need to be perfectly matched to flanking sequences in order to amplify targets
For PCR the sequence of the target does not need to be known; only the sequence of the flankers needs to be known.

primers do NOT have to be perfectly matched to flanking sequences in order to amplify targets.
For PCR the target can be much larger than the primers
Key tools for Recombinant DNA technology:
Restriction Enzymes and DNA Ligase are key tools for Recombinant DNA technology.
Vector(biology):
A vector (in biology) is a DNA molecule that is used to transfer foreign genetic material into another cell)
Good vectors for cloning in E.Coli:
Plasmids(naturally occuring circular DNA in bacteria) and bacteriophage lambda( a virus)
The vector is prepared for accepting the new DNA by:
The vector is prepared for accepting the new DNA by cleaving it at a single specific site with a RESTRICTION ENZYME.
This creates staggard cuts and these cuts are cohesive(sticky-will bond with itself). so.. ANY DNA fragment with the same sticky ends can be inserted in.
Fragments which bond with the plasmid can be made by:
using the same restriction enzyme that was used to open the plasmid DNA
The DNA fragment and the cut plasmid can be joined by using ______, which catalyzes the formation of a phosphodiester bond at a break in a DNA chain.
The DNA fragment and the cut plasmid can be joined together using DNA Ligase, which ____
DNA Ligase requires:
DNA Ligase requires a free 3'-hydroxyl group and a 5'-phosphoryl group

-the chains joined by a ligase must be in a double helix

-ATP and NAD+ are required
Insertional Inactivation:
Insertional Inactivation occurs when a section of DNA is inserted into a Plasmid at a restriction site which interferes with the gene.
pBR322:
this plasmid has an origin of replicatoin and a selectable marker based on ampicillian resistance

aka.. this plasmid can be effected by DNA insertion only.
PCR requires:
PCR requires the primers sequences
(Recombinant DNA)

___________: allow specific DNA to be identified (restriction maps) and manipulated.
Restriction enzymes allow specific DNA to be identified and manipulated
restriction enzymes recognize;
restriction enzymes recognize specific base sequences in a double helical DNA and cleave, at a specific location, both strands of the duplex
Restriction fragments can be separated by
gel elecrophoresis
Southern Blotting:
DNA
Northern Blotting
RNA
DNA sequencing: the precise nucleotide sequences in DNA can determine:
-gene architecture

- controll of gene expression

- protein structure (function)
MUST know for PCR
flanking sequence
Joins DNA fragments and cut plasmids
DNA Ligase
DNA ligase requires
a 3'-OH and 5'phosphoryl group

energy (ATP)
Are descended from a common ancestor:
homologs
paralogs:
same species-different function
orthologs:
different species but similar function
understanding the homology reaveals:
evolutionary history and information about their function
If a substrate has a high kM that means:
the enzyme probably has a low affinity for the substrate

LOWER THE KM THE HIGHER THE AFFINITY FOR THE SUSTRATE
2 evolutionary events are represented by branch points:
specization

genetic duplication
Reverse transcriptase requires:
- all 4 dNTPs

- a primer

-an RNA template
How are sequence allignments made:
Two sequences are alligned to maximize the number of "matches" (base matches). Sometimes gaps are inserted to maximize the number of matches. statistical analysis reveals the significance of the matches by determining the probability that the matches were random or not.
Molecule by which most of the CO2 in the blood is transported through
Bicarbonate
An example of paralogs include:
hemoglobin, myoglobin
The biological role of restriction enzymes in bacteria is to:
cleave foreign DNA
Can be used to add nucleotides to the 3'end of DNA during cDNA synthesis
Terminal Transferase
site of O2 binding:
Heme group
2,3 BPG ___ O2 affinity because
2,3-BPG decreases O2 affinity because it stabilizes th T-state
HbF has a ___ affinity because:
HbF has a higher affinity than Hb because it has a substitution which makes it more likely to bond with oxygen (hist=ser)
The Bohr effect results from formation and breaking of:
ionic bonds
The Bohr Effect:
higher CO2 levels decrease the affinity of Hb for O2 by lowering the pH (increasing H+ atoms from carbonic acid).
Catalyst in Bohr effect:
carbonic anhydrase
-ly charged carbamates form ___ which ___
-ly charged carbamantes form salt bridges which stablize the T-state
A steric inhibitor binds to:
a steric inhibitor binds to the active stite
an allosteric inhibitor binds:
not to the active site
Competitive inhibitor:
binds to substrate at the active site
Competative inhibitor results:

can be overcome by:
Km increases, Vmax not effected

can be overcome by adding more solute
uncompetitive inhibitor:

can be overcome by:
binds to ES complex,

can be overcome by nothing
uncompetitive inhibitor results:
Vmax= lowered

Km= lower
Noncompetitive inhibitor:

can be overcome by:
binds to a site other than the active site

can be over come by nothing
noncompetative inhibitor results:
Vmax=lower
Km= same
myglobin binds;
o2 in muscle, storage
hemeglobin binds:
main oxygn transport
distant evolutionary relationships can be detected through:
substitution matricies
BLAST
basic local allignment search tool
Which structure (3* or 1*) is more conserved? Why?
3 is more conserved than 1 because a proteins 3D shape determins its function. Sometimes substitutions and changes in the 1* chain of amino acids can result in similiar 3Dstructure which whill retain the same functio
divergent evolution:
same ancestor
convergent evolution
different pathways which lead to the same solution (function)
Hglob and Mglob are ___ related to each other
evolutionarly
definition of allosteric enzyme;
when a nonsubstrate molecule inducesa conformational change

when effectsthe affinity of the enzyme for a substrate
Proteolytic enzymes
catalyze the hydrolysis of peptide bonds
the interaction of the enzyme and substrate at the active site promotes the formation of the:
transition state
the active site of an enzyme is the part:
which most greatly lowers the activation energy
mRNA
messenger RNA, template
tRNA
transfer RNA, transfer active amino acids to the ribosome
rRNA
ribosomal RNA, catalytsts for protein synthessi
snRNA
Small Nuclear RNA, splice exons
miRNA
MicroRNA; non coding DNA which bind to mRNA and inhibit their translation
siRNA
Small Interfeering RNA
bind to mRNA and facilitate its degredation; inhibits the expression of genes
RNA polymerase requires:
a template
activated precursors
Mg2+