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129 Cards in this Set
- Front
- Back
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Components of a Nucleotide
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Sugar, Phosphate, Base
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Purines
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Adenine & Guanine
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Pyrimidines
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Cytosine & Thymine
(Uracil replaces Thymine in RNA) |
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Introns
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Transcribed, but cut out of newly synthesized RNA, leaving mature RNA w/ continuous exons
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DNA stability compared to RNA
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RNA is less stable because it has two -OH groups, so it's more prone to hydrolysis.
DNA only has one -OH group, also phosphodiester bridge has a negative charge which repels nucliophilic species, making it more stable |
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NucleoSIDE
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A unit consisting of a base bonded to a sugar
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The four nucleoSides in RNA
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adenOSINE; guanOSINE; cytIDINE; urIDINE
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The four nucleoSides in DNA
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deoxyadenosine; deoxyguanosine; deoxycytidine; thymidine
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NucleoTIDE
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A nucleoSIDE joined to one or more phosphate groups by ester linkages
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DNA polymerase
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catalyzes chain-elongation reaction: nucleophilc attack by 3' -OH terminus of growing chain on innermost phosphorous atom of deoxynucleoside triphosphate
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How does a DNA chain have polarity?
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One end of the chain has a free 5'-OH group (or a 5`-OH group attached to a phosphate,) whereas the other end has a free 3'-OH gropu, neither of which is linked to another nucleotide.
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Directionality that base sequences are written
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5' -> 3'
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How many nucleotides in the human genome in each chain of DNA?
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aprox 3 billion
(divided among 24 chromosomes) |
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How long would a DNA molecule stretch if it were fully extended?
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over a foot
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How far apart are nucleotide bases?
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3.4 angstroms
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Features of Watson-Crick model of DNA
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1. Two helical polynucleotide chains are coiled around a common axis. Chains run in opposite directions.
2. The sugar-phosphate backbones are on the outside and the bases lie on the inside of the helix 3. The bases are nearly perpendicular to the helix axis, and adjacent bases are separated by 3.4A. The helical structure repeats every 34A, and so there are 10bases per turn of the helix. 4. The diameter of the helix is 20A |
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How does the stacking of base pairs contribute to the stability of a double helix?
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1. Double helix is stabilized by the hydrophobic effect (hydrophobic bases cluster in interior of helix) & polar surfaces are exposed to water.
2. Stacked base pairs attract one another through van der Waals forces - small individually, but so many that the net effect is substantial. |
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Semiconservative Replication
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Produces two copies of DNA, each contains one original strand and one newly synthesized strand.
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"Proof" of Semiconservative Replication
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1958: Meselson & Stahl
* labeled parent DNA w/ 15N (heavy nitrogen isotope) to make it denser than normal DNA * after incorperation of heavy nitrogen was complete, bacteria (which was growing the DNA) was transfered to a medium containing only regular nitrogen * let DNA grow: watched successive distribution of each nitrogen. |
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Semiconservative Replication
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.
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Guanine - Cytosine Pairing
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.
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Adenine
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.
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Thymine
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.
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Adenine-Thymine Base Pairing
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.
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Tm
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The melting temperature at which half the helical structure of DNA is lost
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Helicases
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proteins inside the cell which use chemical energy (from ATP) to disrupt helix
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Hypchromism
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Stacked bases in nucleic acids absorb less ultraviolet light than do unstacked bases.
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How do we monitor the melting of nucleic acids
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Measure their absorption of light (maximal at wavelength 260nm)
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Annealing
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The "renaturing" process -> separated complementary strands of nucleic acids spontaneously reassociate to form a double helix when the temperature is below the Tm
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Relaxed Molecule
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A circular DNA molecule without any superhelical turns
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Leading Strand
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Synthesized continuously
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Lagging Strand = Okasaki Fragments
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The lagging strand of DNA is composed of short stretches of RNA primer plus newly synthesized DNA approximately 100-200 bases long (the approximate distance between adjacent nucleosomes)
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DNA polymerase
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* catalyzes the step-by-step addition of deoxyribonucleotide untis to a DNA chain
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4 Features of DNA synthesis
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1. The rxn requires all four activated precursors
- dATP, dGTP, dCTP, and TTP, as well as Mg2+ ion 2. New chain is assembled directly on preexisting DNA template 3. Requires a primer 4. DNA polymerase can correct mistakes -- remove mismatched nucleotides |
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Primer
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Nucleic acid strand -- serves as starting point for DNA replication.
Required because most DNA polymerases cannot begin synthesizing a new DNA strand from scratch -- can only add to an existing strand of nucleotides. In nature, primer is short strand of RNA (which is later replaced w/ DNA) |
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DNA polymerase
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* Synthesize DNA in the 5' to 3' direction.
* Must start w/ primer: can only add a nucleotide onto a preexisting 3'-OH group. |
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Apoenzyme
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An enzyme that requires a cofactor but does not have one present. Example: trp repressor when Trp is absent.
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mRNA
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Encodes and carries info from DNA during transcription to sites of protein synthesis, where they are translated to yield a gene product.
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mRNA "lifecycle"
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The "life cycle" of an mRNA in a eukaryotic cell. RNA is transcribed in the nucleus; once completely processed, it is transported to the cytoplasm and translated by the ribosome. At the end of its life, the mRNA is degraded
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small nuclear RNA (snRNA)
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molecules participate in the splicing of RNA exons
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signal recognition particle
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an RNA-protein complex in the cytoplasm that helps target newly synthesized proteins to intracellular compartments and extracellular destinations
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micro RNA (miRNA)
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class of small (~21 nucleotides) non-coding RNAs that bind to complementary mRNA molecules and inhibit their translation
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small interfering RNA (siRNA)
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class of small RNA molecules that bind to mRNA and facilitate its degradation.
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telomerase
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An enzyme that maintains the telomeres (ends) of chromosomes during DNA replication. Contains RNA.
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ribosomal RNA (rRNA)
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Major component of ribosomes. Catalyst for protein synthesis.
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Transcription
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The synthesis of RNA from a DNA template. Catalyzed by RNA polymerase.
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The reaction of RNA elongation:
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(RNA)n + ribonucleoside triphosphate <-> (RNA)n+1 + PPi
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Required componenets of RNA polymerase
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1. TEMPLATE: preferably double stranded DNA (single stranded can also work). RNA won't work
2. ACTIVATED PRECURSORS: all four ribonucleoside triphosphates (ATP, GTP, UTP, CTP) 3. A DIVALENT METAL ION: either Mg2+ or Mn2+ |
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Dispersive DNA Replication
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parental and daughter material are mixed on each strand
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Conservative DNA Replication
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Two parent strands stay together, and two daughter strands stay together
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Three DNA polymerases
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1. DNA polymerase delta - synthesizes leading strand.
2. DNA polymerase alpha - synthesizes lagging strand. 3. DNA polymerase epsilon: questionable function |
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Synthesis of DNA lagging strand
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1. Primase binds to a signal on the lagging strand that is uncovered by the action of the rep protein.
2. Primase makes a piece of RNA, which serves as a primer for a new DNA polymerase III molecule. 3. Primase moves down the lagging strand, and does this again. 4. The result is a series of short pieces of DNA, each with a RNA primer at the 5' end. These are called OKAZAKI FRAGMENTS. |
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Postreplicative Repair
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Removes errors missed by polymerase proofreading (known to occur in prokaryotes).
1. Double stranded DNA is methylated. This methylation is involved in gene expression, and it takes time for the methylation enzymes to carry out the normal methylation of a new strand of DNA. 2. The mismatched repair system detects distortions caused by any mismatched bases. ○ The mismatched base is excised on the new (presumably defective) strand, which is identified by its lack of methylation. ○ DNA polymerase I fills in the gap. ○ DNA ligase closes the last phosphodiester link. 3. This pattern of repair is called excision repair, and is general, but different types of damage involve different enzyme systems to excise the damaged DNA. |
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tRNA
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The different tRNAs that accept a given amino acid are called isoacceptors. Each carries only one amino acid.
Structure: a "cloverleaf" consisting of a stem and three loops. A small "extra arm" may also exist. The anticodon loop contains a triplet that · base pairs to mRNA during protein synthesis. This triplet is called the anticodon. · plays a role in specifying which amino acid becomes attached to the tRNA The stem ends in the sequence ...CCA, which is the attachment site for the amino acid. It contains additional determinants of which amino acid is attached to the tRNA tRNA contains many unusual bases, which arise by modification after transcription. |
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RNA Synthesis
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1. initiation at a specific site
2. elongation 3. termination at a specific site |
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Prokaryotic transcription
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(E. coli) was studied first, and is simpler and better understood. E. coli RNA polymerase is a multisubunit enzyme, with different functions ascribed to different subunits. The subunits include the following.
1. 2 alpha -- initiation 2. beta -- phosphodiester bond formation 3. beta' -- binds DNA template These four subunits are the core enzyme; they alone will carry out transcription, but cannot initiate rapidly at specific sites. 4. sigma -- recognizes the promoter -- binding specificity. The core enzyme plus sigma factor is called the holoenzyme. 5. omega -- unknown function |
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promotor for eukaryotic transcription
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1. TATA box, about 25 bp upstream of the start site. [consensus: TATA(AT)A(AT)]
2. CAAT box, somewhat further upstream, not always present. [consensus: GC(TC)CAATCT] 3. Various factors bind to these and other important sites, and the DNA probably folds so all these proteins can interact with each other and the RNA polymerase they are supposed to control. |
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RNA polymerase I
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· S factor is required for initiation of the rRNA precursor synthesis by RNA polymerase I. This is reminiscent of the role of sigma factor in prokaryotes.
Cleavage of the unused sequences from the primary transcript results in the formation of the final rRNA species. |
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RNA polymerase II
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Makes the precursor of mRNA, which is subsequently modified in the nucleus before transport to the cytoplasm.
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Exon
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expressed information, which is retained in the mature mRNA
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Alternate splicing
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Sometimes nonconsecutive exons are joined, and/or some exons are left out. When this occurs, the different splicing patterns are found specifically in different tissues. Different useful proteins arise in different tissues in this manner from the SAME gene.
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RNA polymerase III
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Makes the precursor of 5S rRNA and tRNA.
· Cleavage of unnecessary sequences from the primary transcript is as before. · In maturation of tRNA 1. Sequences are removed from the ends of the final product and from the interior of the final product 2. A CCA sequence is added to the 3' end. 3. Bases are modified to produce the unusual bases found in tRNA. |
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Polyadenylation
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Covalent linkage of a polyadenylyl moiety to a messenger RNA molecule -- occurs immediately after transcription of DNA into RNA.
* Mechanism by which most mRNA molecules are terminated at their 3' ends -- aids in stability by protecting it from exonucleases * Important for transcription termination, export of the mRNA from the nucleus, and translation. |
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Anti-sense mRNA
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During transcription, double stranded DNA produces mRNA from the sense strand; the other, complementary, strand of DNA is termed anti-sense. Anti-sense mRNA is an RNA complementary in sequence to one or more mRNAs. In some organisms, the presence of an anti-sense mRNA can inhibit gene expression by base-pairing with the specific mRNAs. In biochemical research, this effect has been used to study gene function, by simply shutting down the studied gene by adding its anti-sense mRNA transcript. Such studies have been done on the worm Caenorhabditis elegans and the bacterium Escherichia coli. This plays a part in RNA interference.
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High Performance Liquid Chromatography (HPLC)
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In column chromatography the smaller and more tightly packed a resin is the greater the separation capability of the column. In gravity flow columns the limitation column packing is the time it takes to pass the solution of proteins through the column. HPLC utilizes tightly packed fine diameter resins to impart increased resolution and overcomes the flow limitations by pumping the solution of proteins through the column under high pressure. Like standard column chromatography, HPLC columns can be used for size exclusion or charge separation. An additional separation technique commonly used with HPLC is to utilize hydrophobic resins to retard the movement of nonpolar proteins. The proteins are then eluted from the column with a gradient of increasing concentration of an organic solvent. This latter form of HPLC is termed reversed-phase HPLC.
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What is a chromatosome?
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A nucleosome plus a fifth histone (H1) which holds the structure together.
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What is a nucleosome?
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A nucleosome consists of four histone molecules (H2A, H2B, H3 and H4) with a strand of double helical DNA wrapped around them.
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What is a histone?
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The histones are proteins which are
○ highly conserved across species lines (implies great importance of the structure). ○ basic -- contain lots of arg and lys (pos chg), which interact with DNA via salt bridges. |
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What is a chromasome?
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One very large DNA molecule -- linear in eukaryotes. It has three essential elements.
1. centromere (spindle attachment site during cell division) 2. telomeres (ends) 3. one or (in eukaryotes) many origins of replication (Autonomously Replicating Sequences). If the DNA were very large, and there were only one origin of rep, it would take too long to complete the process. |
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RNA is readily hydrolyzed by alkali, whereas DNA is not. Why?
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The 2'-OH group in RNA acts as an intramolecular nucleophile. In the alkaline hydrolysis of RNA, it forms a 2'-3' cyclic intermediate.
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What is the role of reverse transcriptase ribonuclease activity?
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The ribonuclease serves to degrade the RNA strand, a necessary step in forming duplex DNA from the RNA-DNA hybrid
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Denisty of cesium chloride
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Similar to that of DNA. Therefore can be used in density equilibrium sedimentation.
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What does color show in an indirect ELISA?
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Amount of an antibody specific to an antigen
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What does color show in a sandwich ELISA?
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Indicates the quantity of antigen.
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What would happen if serum were omitted from the ELISA, but all other steps remained the same and were performed properly?
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* Anti-human Ig-conjugate would not bind and be washed away.
* The O.D. values would be nearly the same as the assay control. |
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What would happen if the anti-human Ig-conjugate were not washed free of the well before the substrate was added?
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All wells would show uniform overdevelopment due to unbound and excess anti-human Ig enzyme conjugate.
Since the enzyme which acts on the substrate is in excess, it would turn all the wells a uniform color whether they were truly positive or not. |
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What does color show in a sandwhich ELISA?
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The rate of color formation is proportional to the amount of antigen
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What does color show in an indirect ELISA?
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The rate of color formation is proportional to the amount of specific antibody
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Holoenzyme
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Active form of enzyme + necessary cofacotr.
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What is the length of DNA in E. coli? (in nucleotides)
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Single DNA molecule consisting of two chains of 4.6 million nucleotides each.
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What is the length of DNA in a virus? (in nucleotides)
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5100 nucleotides
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What is the length of DNA in an average person?(in nucleotides)
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3 billion nucleotides divided among 24 chromosomes.
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what is the bond between 5'carbon in the sugar, and the base?
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B-glycosidic linkage
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What is the diameter of the double helix?
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20A
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What is hypochromism?
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The name of the fact that stacked bases in nucleic acids absorb less ultraviolet light than unstacked bases.
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What do helicases do?
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Since heat is not used to disrupt the double strand of DNA (for They use chemical energy from ATP to disrupt the helix (when double
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How can we break a double helix in the lab? (three ways)
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1. Heat
2. add acid 3. add alkali to ionize base |
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What is the definition of melting temperature for DNA
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the temperature at which half the helical structure is lost.
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What does it mean for a DNA molecule to be supercoiled? Why are some DNA molecules supercoiled?
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the axis of the double helix is twisted (supercoiled) into a superhelix.
1. more compact 2. may hinder or favor the capacity of the double helix to unwind -> affect interactions between DNA and other molecules. |
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What is a stem-loop structure?
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Formed from single-stranded DNA and RNA molecules. Formed when two complementary sequences within a single strand come together to form double-helical structure.
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What is PPi?
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pyrophosphate ion
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What is reverse transcriptase?
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A viral enzyme that copies RNA (from the single-stranded RNA molecule that enters the cell) into DNA.
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Which direction does chain elongation of DNA go?
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5' to 3'
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What is a virus?
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A genetic element enclosed in protein coats - can move from one cell to another but are not capable of independent growth.
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What is an RNA-directed RNA polymerase?
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An RNA polymerase that takes directions from an RNA template, to copy viral RNA.
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What is a retrovirus?
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Genetic information flows from RNA to DNA (rather than DNA to RNA).
* contain 2 copies of sngle-stranded RNA molecule * RNA enters cell, copied into DNA by reverse transcriptase. * results in double-helical DNA version of viral genome -- can be encorperated into normal cellular DNA. |
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What is the "flow of genetic information" during gene expression?
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DNA -> (transcription) -> RNA -> (translation) -> Protein
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What is transcription?
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RNA is made from a DNA template by RNA polymerase. The beginning of the unit to be transcribed is a promoter site that specifically binds RNA polymerase (TATA box)
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How long is the distance between 3.6 residues?
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0.54nm
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What are the advantages of having introna and exons?
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* new proteins may arise in evolution by rearranging exons
* potential for generating a series of related proteins by splicing a nascent RNA transcript in different way |
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Alternative Splicing
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generates mRNAs that are templates for different forms of a protein: easy way of forming a set of proteins that are variations of a basic motif without requiring a new gene for each protein
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Shine-Dalgarno sequence
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In bacteria, purine-rich sequence which has a complementary sequence with a rRNA molecule -- procedes the initiation codon
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Spliceosome
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assemblies of proteins and small RNA molecules which facilitate splicing. Recognizes signals in the nascent RNA that specify the splice site -- usually begin with GU and end with AG
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Restriction Enzyme (endonuclease)
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Cleave foreign DNA molecules - cell's own DNA is not degraded because the sites recognized by its own restriction enzymes are methylated. Cleavage sites are almost always palindromic.
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3 Features of spliceosomes
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1. are composed of RNA and proteins
2. recognize RNA sequences for the removal of introns 3. can produce differnet mRNA molecules by splicing at different sites. |
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When might nucleic acids form non Watson-Crick base pairs?
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Single stranded RNA can bond with itself: H-bonds can be made with non-standard pairs. These bonds are often stabilized by metal ions
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Hemoglobin oxygen binding curve: 5 causes that shift it to the right
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CADET, FACE RIGHT!
(C)02 (A)cid 2,3(D)PG (= 2,3,BGG) (E)xercise (T)emperature |
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mnemonic for remembering what different blots measure
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SNOW DROP:
Southern DNA Nourthern RNA OOOOO Western Protein |
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Eukaryotic Polymerases (4)
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Eukaryote polymerases:
Alpha - lAAAAgging strand Beta & epsilon involved in DNA repair Delta - leaDDDDing strand Gamma- the only one left is for mitochondrial DNA |
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How to remember the 3 RNA polymerases
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1)RNA poly ONE is the Real ONE
poly I--> rRNA 2) mRNA--> choose m for Middle number RNA poly II--> mRNA 3) Three goes with T RNA polymerase III-->tRNA |
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How can you use a DNA restriction enzyme (endonuclease) and RNA polymerase to locate a promoter site?
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First incube double stranded DNA with RNA polymerase - it will bind tightly to promotor site; then add DNA endonuclease - it will degrade anything that isn't bound. Wash away and sequence what's left.
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What bacterial enzyme is needed to initiate viral infection when T2 DNA first enters cell?
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Because only DNA and no protein enters the cell, synthesis of viral directed protein cannot begin until mRNA is made.
-- transcription of T2 DNA must be carried out by bacterial RNA polymerase using ribonucleoside triphosphates from bacteria. |
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Does RNA polymerase have the ability to correct mistakes?
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NO: not sharing genetic material to progeny, only making proteins - malformed proteins can be gotten rid of by the cell later, if nec.
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Does DNA polymerase have the ability to correct mistakes?
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YES
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How many bases are there per turn in a DNA double helix?
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10 (structure repeats every 34A, 3.4A per base)
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What is the strength of a H-bond in DNA double helix?
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4-21 kJ/mol
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Does the absorbance of a DNA solution increase or decrease when the double helix is melted into a single strand?
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INCREASES (known as hypochromism) -- the curve will look the same, just be shifted upwards (where y-absorbance and x-wavelength)
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What does polynuclease phosphorylase do?
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It polymerizes ribonuclease diphosphates (NDP) to form RNA and Pi
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In Hb, what would a mutation from leu to arg do to a-helix?
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Destabilizes: substituting hydrophobic residue for hydrophilic (which is probably in the interior of the protein)
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In Hb, what would a mutation from leu to val do to a-helix?
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No effect (conservative replacement of similar hydrophobic residues)
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In Hb, what would a mutation from leu to pro do to a-helix?
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Destabilizes: proline does not allow for rotation about peptide bond & no hydrogen is available for H-bonding
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In Hb, what would a mutation from leu to gly do to a-helix?
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Destabilizes: very flexible residue, acts as swivel, disrupts helix.
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In Hb, what would a mutation from leu to ala do do to a-helix?
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No effect (conservative replacement of similar hydrophobic residues)
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Are glycines common in a helix?
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No, they are very flexible, so they can act like a swivel and disrupt the helix.
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What does a Hill Plot show?
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linear plot that shows cooperativity of oxygen binding to hemoglobin
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What is the Hill coefficient
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Measure of cooperativity of oxygen binding.
** 2.8 ** |
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What is the slope of the Myoglobin Hill plot?
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1 -- linear; only one binding site, no "cooperation"
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What is the slope of the Hemoglobin Hill plot?
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2.8 -- sigmoidal -- as binding increases, so does slope, until there are few binding sites left, and slope tapers slightly.
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(4)Parameters of the Concerted Model explaining cooperation in Hemoglobin binding of oxygen
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1. number of binding sites in the protein
2. ratio of teh concentration of T and R states in the absence of bound ligands 3. affinity of sites in proteins in the R state for ligand binding 4. a measure of how much more tightly subunits in proteins in the R state bind ligands compared to subunits in the T state |
