Term
| Experimental Evidence shows that DNA was |
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Definition
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Term
| 4 Characteristics of Genetic Material |
|
Definition
1. Replication
Fully copied and separated into daughter cells during cell division |
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Term
| 4 Characteristics of Genetic Materia |
|
Definition
2. Storage of information
Encode all the information needed for all cells for the organism |
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Term
| 4 Characteristics of Genetic Materia |
|
Definition
3. Expression of information
Decoding of the information into functional molecules |
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Term
| 4 Characteristics of Genetic Material |
|
Definition
4. Variation by mutation
Vehicle for adaptation |
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Term
| Expression of information |
|
Definition
| DNA --> Transcription --> RNA --> Ribosome --> Translation --> Protein |
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Term
|
Definition
Showed that DNA was the genetic material
Bacteriophage DNA alone was sufficient to make new bacteriophage virions
Study The Experiment |
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Term
| Building Blocks of Nucleic Acids |
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Definition
DNA and RNA are composed of similar building blocks
Similarity is essential to information flow
Differences are also essential to the division of labor
Chemistry of DNA and RNA is necessary to understand structure |
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Term
Nucleotides
3 Essential Components |
|
Definition
1. Phosphate group
2. Pentose sugar
Two types: ribose (RNA) and deoxyribose (DNA)
3. Nitrogenous base
Two types of base: purines and pyrimidines
Purines: adenine and guanine
Pyrimidines: Thymine (DNA only), cytosine, and uracil (RNA only) |
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Term
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Definition
| No oxygen on the # 2 Carbon |
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Term
|
Definition
Side: Has a pentose w/ a Nitrogenous base.
Tide: The Same, but with a phosphate group |
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Term
| Nucleoside Triphosphate can be added to DNA bc |
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Definition
| It has 3 Phosphate Groups |
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Term
| ATP and GTP are important molecules in cellular energetics. |
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Definition
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|
Term
| Hydrolysis of ATP or GTP to ADP or GDP and inorganic phosphate releases a large amount of energy |
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Definition
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Term
| Formation of Polynucleotides |
|
Definition
| Two mononucleotides are linked by a phosphodiester bond between the phosphate group of one and the sugar of another |
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Term
| Formation of Polynucleotides |
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Definition
| The phosphate group connects the 3’ carbon of one sugar to the 5’ carbon of the next |
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Term
| Formation of Polynucleotides |
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Definition
| Thus, each strand of nucleic acid has a 3’ end and a 5’ end |
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Term
| Formation of Polynucleotides |
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Definition
| You can only add new nucleotides to the 3' end |
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Term
| Phosphate binds to its own at |
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Definition
| 5' end, and to a new at the 3' end |
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Term
|
Definition
Two chains
Antiparallel (3’-5’, 5’-3’)
Right-handed turn
Stacked bases in center
Bases are paired: G-C, A-T
10 bases per turn
Alternating major and minor grooves |
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Term
|
Definition
| The thumb follows (of the right hand) follows the backbone |
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Term
|
Definition
| Pyramidines and Purines bind togther |
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Term
|
Definition
| Turns go from Major groove to minor groove |
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Term
|
Definition
Watson, Crick, and Franklin observed B-form DNA
Biologically relevant form (what I showed you) |
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Term
|
Definition
However, very dehydrated DNA forms A- or C-DNA
Less compact that B-DNA, with 9 and 9.3 bases per turn, respectively |
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Term
|
Definition
| Also, D-DNA and E-DNA are found in helices lacking guanine |
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Term
|
Definition
| P-DNA forms when B-DNA is stretched out |
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Term
|
Definition
Z-DNA forms when helices of only G-C are made
May form in vivo at stretches of high G-C content or during certain cellular processes
Forms left-handed helix, others are all right-handed |
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Term
|
Definition
| RNA has ribose, not deoxyribose |
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Term
|
Definition
RNA and DNA have three of the same bases
RNA has uracil, DNA has thymine – both pair with adenine
However, RNA is found as a single strand biologically |
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Term
|
Definition
Except:
Some portions of an RNA strand can fold back on themselves into double-stranded regions
Some viruses have double-stranded RNA genomes
Biologically, RNA is single stranded but it can fold back on itself for stability (Usually in Eukaryotes) |
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Term
|
Definition
| DNA and other molecules are often described and named based upon their sedimentation behavior. |
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Term
|
Definition
| A mixture of nucleic acids (or other molecules) is loaded on top of a tube containing concentration gradient |
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Term
|
Definition
| The tubes are spun in a high speed centrifuge; the nucleic acids migrate through the gradient at different rates |
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Term
|
Definition
| The gradient solution is then eluted out in small fractions and examined further |
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Term
|
Definition
Sedimentation equilibrium centrifugation
Sedimentation velocity centrifugation |
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Term
| Sedimentation equilibrium centrifugation |
|
Definition
Uses a density gradient
Molecules of different, if similar, densities can be separated
Also, can determine the base composition of dsDNA
G-C pairs have 3 bonds, so more dense than A-T pairs with 2 bonds |
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Term
| Sedimentation velocity centrifugation |
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Definition
Migration based on shape and weight
Seen in particular with proteins
Can be used to determine molecular weight of molecules |
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Term
| Denaturation and Renaturation |
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Definition
Under heat or other stress, DNA can denature or melt
H bonds break, but covalent bonds remain in the backbone
***Dont confuse with Protein denaturation***** |
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Term
| Denaturation and Renaturation |
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Definition
| DNA loses it’s function when it is denatured completely |
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Term
| Denaturation and Renaturation |
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Definition
| DNA loses it’s function when it is denatured completely |
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Term
| Denaturation and Renaturation |
|
Definition
| However, DNA can renature under the correct circumstances |
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Term
| Denaturation and Renaturation |
|
Definition
| UV light absorption can be used to detect denaturation of a segment of DNA |
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Term
| Denaturation and Renaturation |
|
Definition
| Also, the G-C vs A-T content can be measured |
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Term
| Denaturation and Renaturation |
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Definition
| It takes more energy (heat) to break 3 hydrogen bonds that it does to break 2 |
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Term
| Denaturation and Renaturation |
|
Definition
| Melting point (Tm) is the temperature at which a piece of DNA will denature |
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Term
| Denaturation and Renaturation |
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Definition
| Higher Tm = more base pairs that have to be broken (G-C) |
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Term
|
Definition
| When cooled properly, renaturation can occur (H Bonds reform) |
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Term
|
Definition
The ability of DNA to denature and renature is essential to it’s function!
When in double strand confirmation, the bases are protected, otherwise information can be accessed |
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Term
| Denaturation and Renaturation |
|
Definition
| If DNA could not be opened and separated into individual strands, replication and transcription could not occur |
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Term
|
Definition
Two similar pieces of DNA (or DNA and RNA) from different sources can be joined under the correct conditions
Shows similarity in genes between organisms
So genes similar throughout organisms, some only found in certain species |
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Term
| Polymerase Chain Reaction (PCR): |
|
Definition
Used to amplify DNA sequences in order to visualize them
Use small pieces of single-stranded DNA called primers
Will use this technique in lab |
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Term
|
Definition
- Uses Argose Gel
- Diff Pore Sizes
- Uses charge to more + or - DNA
- *** Smaller DNA migrates down farther *** |
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Term
|
Definition
| Molecules move at a rate inversely proportional to their length. The longer they are, the slower they move |
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Term
| How does the cell make more DNA for the next daughter cell? |
|
Definition
Three possible ways:
Conservative
Dispersive
Semiconservative |
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Term
|
Definition
|
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Term
|
Definition
|
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Term
|
Definition
| Cytosine and Guanine and Uracil (RNA) |
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Term
|
Definition
We have learned that in replication, dsDNA separates, or denatures
Where the DNA separates is called the origin of replication |
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Term
| Meselson-Stahl Experiment |
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Definition
Used bacteria to show which form of replication actually occurred
Grew E. coli in culture labeled with 15N
Then bacteria were grown in media with 14N
The DNA was spun through a gradient, where the heavier 15N DNA would be found at the bottom, DNA with both found in the middle, and DNA with 14N found near the top |
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Term
|
Definition
| If conservative replication, there would be two fractions, top and bottom |
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Term
|
Definition
| If dispersive, a mixed population in the middle |
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Term
|
Definition
| If semiconservative, a mixed population in the middle and a group at the top that grew each generation |
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Term
|
Definition
| When the DNA is opened and replication occurs, this creates a replication fork |
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Term
|
Definition
| The replication fork moves along the DNA, opening further along the DNA, continuing replication |
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Term
|
Definition
A replicon is the segment of DNA replicated after one initiation event at a single origin
Replicon: Replicated DNA after 1 iniation |
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Term
|
Definition
| One origin of replication at oriC |
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Term
|
Definition
| Bi-directional replication in both directions from oriC |
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Term
|
Definition
| One replicon, encompassing the entire genome |
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Term
|
Definition
| Replication stops at the ter (termination) site |
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Term
|
Definition
|
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Term
|
Definition
| Multiple origins of replication, thus multiple replicons |
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Term
|
Definition
| is more than one chromosome, must have more than one replicon |
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Term
|
Definition
| Replication termination is more complicated in eukaryotes |
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Term
|
Definition
| Arthur Kornberg discovered that enzymes are involved in replication |
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Term
|
Definition
| DNA polymerase I was shown to direct DNA synthesis outside of the cell if only a DNA template and deoxyribonucleoside triphosphates (dNTPs) were present |
|
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Term
|
Definition
| Only dNTPs with the 3 phosphates attached would lead to DNA synthesis |
|
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Term
|
Definition
| All 4 (A, T, C, G) must also be present |
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Term
|
Definition
| Used as a guideline to create new DNA |
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Term
|
Definition
|
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Term
|
Definition
|
|
Term
| Two phosphate groups are removed and the new dNTP is covalently bonded to the 3’ end of the chain |
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Definition
|
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Term
|
Definition
| DNA pol I was shown to have high fidelity (accuracy) synthesizing ssDNA and it also had exonuclease activity, meaning it could degrade DNA |
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Term
|
Definition
| Further studies showed that lacking DNA pol I did not stop replication, but that DNA repair was inhibited |
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Term
|
Definition
| Thus, DNA pol I cannot be the only polymerase and it must have dual activity |
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Term
| DNA pol II, III, IV, and V |
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Definition
| DNA pol I, II, and III can all elongate DNA but cannot initiate synthesis of new DNA |
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Term
| DNA pol II, III, IV, and V |
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Definition
|
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Term
| DNA pol II, III, IV, and V |
|
Definition
| Can backtrack and remove bases 3’-5’ |
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Term
| DNA pol II, III, IV, and V |
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Definition
| DNA pol I has 5’ -3’ exonuclease activity and is found in higher concentration |
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Term
|
Definition
| DNA pol III is important of 5’-3’ polymerization of the DNA strand (adding bases) |
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Term
|
Definition
Also, its 3’-5’ exonuclease activity allows it to remove incorrect bases
Called proofreading |
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|
Term
|
Definition
DNA pol I is thought to use its 5’-3’ exonuclease activity to remove the primer needed at the beginning of replication and to fill in the gap left by removal
Also involved in DNA repair |
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Term
|
Definition
DNA pol II, IV, and V are involved in repairing DNA damaged by external forces
UV light, oxygen radicals, etc |
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Term
|
Definition
| DNA pol II is activated when replication is disrupted at the replication fork |
|
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Term
|
Definition
| Not just one peptide, active form is a holoenzyme of multiple subunits |
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Term
|
Definition
|
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Term
|
Definition
| α, ε, and θ subunits make up the core enzyme responsible for the polymerization activity |
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Term
|
Definition
| α is responsible for nucleotide addition |
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Term
|
Definition
| Ε possesses the 3’-5’ exonuclease acitivty |
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Term
|
Definition
| One DNA pol III has TWO core enzymes combined into a dimer |
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Term
|
Definition
| Second group of subunits (γ, δ, δ’, χ, ψ) make up the γ (gamma) complex |
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Term
| DNA pol III γ (gamma) complex |
|
Definition
This complex is involved in loading the enzyme onto the template DNA at the replication fork
This step requires energy through hydrolysis of ATP |
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Term
| DNA pol III γ (gamma) complex |
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Definition
|
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Term
|
Definition
| β subunits serves as a clamp, keeping DNA pol III on the template during replication |
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Term
|
Definition
| τ is needed for dimerization of two core polymerases at a replication fork |
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Term
|
Definition
Unwind the Helix
Initiation of DNA synthesis with RNA primer
Continuous and Discontinuous synthesis of antiparallel DNA strands
Concurrent Synthesis on Leading and Lagging strands
Integrated Proofreading and Error Correction |
|
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Term
|
Definition
| oriC is characterized by sequences of 9 and 13 bases |
|
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Term
|
Definition
| Several subunits of a protein DnaA bind to the 9-mers, causing initial DNA unwinding |
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|
Term
Unwinding the DNA helix
helicases |
|
Definition
DnaB and DnaC bind next, further stabilizing and opeing the DNA helix
helicases |
|
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Term
|
Definition
| DNA conformation is stabilized by single-stranded binding proteins (SSBPs) |
|
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Term
|
Definition
| unwinding causes supercoiling |
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Term
|
Definition
| DNA gyrase (DNA topoisomerase) keeps supercoiling under control |
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Term
|
Definition
| Makes single-or double-stranded cuts in the DNA and moves the DNA back around itself to relax the tension; then the strands are resealed |
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Term
| Initiation of DNA Synthesis |
|
Definition
| DNA pol III needs a 3’ end to add new nucleotides to because it can’t start DNA synthesis de novo |
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Term
|
Definition
| generates an RNA primer of 10-12 nucleotides that DNA pol III uses to begin DNA synthesis |
|
|
Term
Initiation of DNA Synthesis
DNA pol I |
|
Definition
| removes the primer after the replication fork moves |
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Term
| Continuous and Discontinuous DNA Synthesis |
|
Definition
| The 2 strands in DNA double helix are antiparallel |
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Term
| Continuous and Discontinuous DNA Synthesis |
|
Definition
| Replication fork travels down the DNA in one direction, but DNA pol III can only add bases 5’-3’ |
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Term
| Continuous and Discontinuous DNA Synthesis |
|
Definition
| Addition of bases goes in opposite directions |
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Term
| Continuous and Discontinuous DNA Synthesis |
|
Definition
| One strand is always behind the other |
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Term
|
Definition
| Leading strand moves in the same direction as the replication fork |
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Term
|
Definition
| is polymerized opposite of the replication fork |
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Term
| Continuous and Discontinuous DNA Synthesis |
|
Definition
As the replication fork moves, a new primer is needed for the lagging strand
Nucleotides cannot be added to the 5’ end of the previous primer |
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Term
|
Definition
|
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Term
|
Definition
|
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Term
|
Definition
| The many short fragments of DNA on the lagging strand |
|
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Term
| DNA pol I removes the primers from the Okazaki fragments via |
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Definition
|
|
Term
| Ligase joins the DNA fragments togther by |
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Definition
|
|
Term
| ___________ also fills in the gaps left by primer removal |
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Definition
|
|
Term
| Concurrent Strand Synthesis |
|
Definition
| DNA pol III dimer can replicate both strands if the lagging strand is looped to allow movement in the same direction as the replication fork |
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Term
| Concurrent Strand Synthesis |
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Definition
| As new Okazaki fragments are formed, new loops are made further down stream |
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Term
|
Definition
| Proofreading and error correction occur during replication |
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Term
|
Definition
| DNA polymerases have proofreading activity and correct errors with 3’-5’ exonuclease activity |
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Term
|
Definition
Multiple replication origins
Called replication bubbles |
|
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Term
|
Definition
| Each bubble offers two replication forks |
|
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Term
|
Definition
| Need multiple origins because more DNA and replication is slower |
|
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Term
|
Definition
| 20-80 adjacent origins are replicating, then 20-80 more until entire genome is complete |
|
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Term
|
Definition
| In eukaryotes, how origins are marked. These are stretches of DNA that are conserved throughout species |
|
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Term
| origin recognition complex (ORC) |
|
Definition
| The consensus sequences are recognized by this group of proteins |
|
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Term
| origin recognition complex (ORC) |
|
Definition
When the ORC is activated, initiation occurs; including DNA unwinding
A-T rich regions are present at origins; they are easier to melt than G-C rich regions |
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Term
| Eukaryotic DNA Polymerases |
|
Definition
| four replicative polymerases (involved in replication) |
|
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Term
|
Definition
| Pol α, δ, and ε are required for nuclear DNA replication |
|
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Term
|
Definition
| Pol β and ζ are involved in DNA repair |
|
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Term
|
Definition
| Pol γ is needed for replication of mitochondrial DNA |
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Term
| Eukaryotic DNA Polymerases |
|
Definition
| Pol α and δ are the major enzymes in DNA synthesis |
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Term
| Eukaryotic DNA Polymerases |
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Definition
| Pol α generates the RNA primer needed for initiation |
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Term
| Eukaryotic DNA Polymerases |
|
Definition
| Once the primer is in place, Pol δ replaces Pol α: called polymerase switching |
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Term
| Eukaryotic DNA Polymerases |
|
Definition
Pol δ elongates from the primer
It also has 3’-5’ exonuclease activity |
|
|
Term
| Eukaryotic DNA Polymerases |
|
Definition
| Pol ε is thought to switch with Pol δ in order to continue elongation |
|
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Term
| Eukaryotic DNA Polymerases |
|
Definition
| This mechanism is the same for both leading and lagging strands |
|
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Term
| Replicating Chromosome Ends |
|
Definition
| Because eukaryotic DNA is linear replicating the ends is necessary |
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Term
| Replicating Chromosome Ends |
|
Definition
| The double-stranded ends of the chromosome can resemble damage that the cell might attempt to repair or degrade |
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Term
| Replicating Chromosome Ends |
|
Definition
| Inert (non-coding) sequences called telomeres are added to the ends of chromosomes in order to protect coding DNA from changes through repair |
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Term
|
Definition
Double-stranded DNA runs antiparallel
So, at an end, one strand will have its 5’ end, the other will have 3’ end
The lagging strand (ending in 3’) will have a space that cannot be replicated
A primer can’t be made to cover it and polymerases can’t fill in the gaps |
|
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Term
|
Definition
| If the gap is unfilled and replication is repeated over and over (through many generations), the gap will eventually eat into the coding DNA |
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Term
|
Definition
An enzyme that adds telomere repeats
Telomerase uses a piece of RNA as a template for base addition |
|
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Term
|
Definition
| added to the leading strand Because telomerase can’t add bases to the 5’ end of DNA, only the 3’ end |
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Term
|
Definition
| Pol α and δ can use the new telomeres as a template to fill in the gap made and seal the end of the lagging strand |
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Term
|
Definition
| Viral DNA is not used when packaged inside the virus, only after infecting a cell |
|
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Term
|
Definition
| Bacterial and eukaryotic DNA must be used within its housing |
|
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Term
|
Definition
| Bacterial and eukaryotic DNA must be used within its housing |
|
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Term
|
Definition
| Therefore, packaging of DNA needs to be efficient and reversible |
|
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Term
|
Definition
| In bacteria, DNA is condensed into a part of the cell called |
|
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Term
|
Definition
| Not a true organelle, it is an area of the cell where the chromosome is concentrated |
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Term
|
Definition
DNA-binding proteins
HU and H1 do not compact DNA like eukaryotic histones |
|
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Term
|
Definition
| They are similar to histones in eukaryotes |
|
|
Term
|
Definition
| Supercoiling has been found to facilitate viral and bacterial condensation |
|
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Term
|
Definition
| A DNA molecule that is supercoiled takes up less space in the cell |
|
|
Term
|
Definition
|
|
Term
| One with fewer turns than the relaxed state is |
|
Definition
|
|
Term
| The strain can be alleviated through |
|
Definition
|
|
Term
| Supercoiling restores the |
|
Definition
resting state
number of turns: 18 plus 2 supercoils = 20
If in the opp direction of DNA, = -
If in the same dir of the DNA = + |
|
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Term
|
Definition
| is made in the opposite direction as the turn of the DNA (left supercoil in right-handed DNA) |
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Term
|
Definition
| Two identical molecules with different linking numbers |
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Term
|
Definition
| Enzymes that cleave the DNA, twist or untwist it, then reseal the ends |
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Term
|
Definition
cleave only one strand
Remove negative supercoils |
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Term
|
Definition
cleave both strands
Introduce negative supercoils |
|
|
Term
| Supercoiling and Topoisomerases |
|
Definition
| Both bacteria and eukaryotes have supercoiled DNA and use topoisomerases |
|
|
Term
| Supercoiling and Topoisomerases |
|
Definition
| In particular, eukaryotes need topoisomerases to relieve supercoiling introduced by replication and transcription |
|
|
Term
| Supercoiling and Topoisomerases |
|
Definition
| As the DNA is opened up, helix downstream takes up the energy and forms supercoils |
|
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Term
|
Definition
| release the tension caused by supercoils |
|
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Term
|
Definition
chromatin is DNA wrapped around proteins called histones
Tightly packed state of DNA |
|
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Term
|
Definition
| major role in the structure of chromatin |
|
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Term
|
Definition
Core histones
Linker histones |
|
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Term
|
Definition
Highly conserved
Cow histone H4 and pea histone H4 differ by only two amino acids |
|
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Term
|
Definition
| is found between octomers |
|
|
Term
|
Definition
right handed DNA (-)
Two supercoils are required to relieve the strain |
|
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Term
|
Definition
is an octomer
Two dimers of H2A and H2B
Tetramer of H3 and H4 |
|
|
Term
|
Definition
|
|
Term
|
Definition
| core octomer + linker histone + ~180bp DNA |
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|
Term
|
Definition
| DNA is wrapped around the core octomer 2X in left-handed superhelical turns |
|
|
Term
|
Definition
| Histone is positive, DNA is negative; most likely electrostatic interactions hold them together |
|
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Term
|
Definition
| DNA --->RNA ---> Protein, via transcription and translation |
|
|
Term
|
Definition
| Code is transcribed from DNA to RNA |
|
|
Term
| Characteristics of the Genetic Code |
|
Definition
Linear
using ribonucleotides as “letters” in messenger RNA complementary to the DNA |
|
|
Term
|
Definition
| triplets of 3 ribonucleotides |
|
|
Term
|
Definition
| Each codon codes for one amino acid |
|
|
Term
|
Definition
| each triple codon codes for only one amino acid, the same one each time the triple occurs |
|
|
Term
|
Definition
| most amino acids are coded for by more than one codon |
|
|
Term
| Start (1 codon) and Stop codons (3 codons) |
|
Definition
signal initiation and termination of the protein
This refers to translation, not start and stop of transcription |
|
|
Term
| Code is read and amino acids are added to the growing peptide until |
|
Definition
| a stop is signaled (no “punctuation”) |
|
|
Term
|
Definition
| each nucleotide is read once, as part of only one triplet |
|
|
Term
|
Definition
| nearly all organisms, viruses to humans, use the same basic code for protein synthesis |
|
|
Term
| Triplets and Reading Frame |
|
Definition
| an addition or subtraction of bases can change the reading frame of the message |
|
|
Term
| Triplets and Reading Frame |
|
Definition
addition of 3 bases together does not shift the reading frame
It does, but not for the entire length of the message |
|
|
Term
| Triplets and Reading Frame |
|
Definition
| translation machinery does not “understand” the code, it just adds amino acids coded for three bases at a time |
|
|
Term
| Degeneracy and the Wobble Hypothesis |
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Definition
| There are 64 codons: 61 that code for amino acids, 1 for start, and 3 for stop |
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Term
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Definition
The first two nucleotides in a codon are more critical than the third
mutations in the third codon typically have less effect on the amino acid coded for and therefore, the protein structure |
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Term
| Degeneracy and the Wobble Hypothesis |
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Definition
some amino acids have 2, 3, 4, or more codons
Allows for the RNA to bind to more than one codon.....it saves energy |
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Term
| Degeneracy and the Wobble Hypothesis |
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Definition
| The first two letters often are all that are needed to code for an amino acid |
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Term
| Degeneracy and the Wobble Hypothesis |
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Definition
| This “wobble” allows a single tRNA to bind with more than one codon and still have the correct amino acid added |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
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Definition
| Codes for fmet (formylated methionine) in bacteria and met (unformylated methionine) in eukaryotes |
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Term
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Definition
| AUG is not at the beginning of the transcript, though |
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Term
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Definition
| fmet or met are often cleaved from the amino acid chain after translation |
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Term
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Definition
| UAG, UAA, and UGA all code for a stop in peptide elongation |
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Term
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Definition
| There are no tRNA molecules that have anticodons for these three, so no more amino acids are added to the chain |
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Term
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Definition
| When a nonsense mutation occurs, an inappropriate stop codon is created and the protein is cut short |
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Term
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Definition
| Synthesis of RNA molecules from a DNA template |
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Term
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Definition
| Transcription is the initial step in information flow in the cell |
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Term
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Definition
| Results in mRNA, which is complementary to the DNA template and to the tRNAs that correspond to each codon |
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Term
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Definition
| the intermediate molecules between DNA and protein coded for in the DNA |
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Term
| RNA polymerase works similarly to DNA polymerase, with two main exceptions |
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Definition
Its substrate nucleotides contain ribose instead of deoxyribose
No primer is needed to initiate synthesis |
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Term
| RNA Polymerase Direct RNA Synthesis |
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Definition
| Nucleotides are linked by 5’ to 3’ phosphodiester bonds, like DNA |
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Term
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Definition
| have a singular circular chromosome |
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Term
| The chromosome is centralized into a |
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Definition
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Term
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Definition
| There is no nuclear membrane as with eukaryotes |
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Term
| As soon as a transcript begins to be made |
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Definition
| ribosomes will begin to translate it |
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Term
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Definition
| An mRNA with more than one ribosome translating it |
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Term
| As soon as a transcript begins to be made |
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Definition
This allows transcription and translation to complete within minutes
Versus hours or longer in eukaryotes
Important because bacteria must be able to adapt quickly to their environment |
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Term
| DNA is organized into genes, which code for proteins or RNAs |
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Definition
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Term
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Definition
| In order to transcribe a gene, a region upstream (5’) |
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Term
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Definition
| This region is recognized by the RNA polymerase and helps to align the enzyme for proper transcription |
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Term
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Definition
| When more than one gene are located together and transcribed from the same promoter, |
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Term
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Definition
| While not all identical, promoters have regions |
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Term
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Definition
| A consensus sequence is not base-per-base the same for every promoter, but some bases are typically located at the same position relative to the translational start site |
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Term
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Definition
| The closer a region is to the consensus sequence, the stronger the interaction with the RNA polymerase |
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Term
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Definition
Two portions make up
Core enzyme
Sigma factor |
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Term
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Definition
| – Catalyzes polymerization, high affinity for most DNA, needs sigma factor for specific binding |
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Term
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Definition
| involved in promoter recognition, interact with -10 and -35 boxes, most commonly used is σ70, alternative factors with different consensus sequences are sometimes used |
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Term
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Definition
Initiation
Elongation
Termination |
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Term
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Definition
Closed promoter complex
Open promoter complex
Promoter clearance |
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Term
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Definition
Holoenzyme binds to -35 and -10 boxes
“Closed” means the DNA is still double-stranded, so this step is reversible |
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Term
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Definition
Holoenzyme undergoes structural change, ~18bp of DNA around +1 is melted
Generally irreversible
Transcription begins; no primer needed, only NTPs |
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Term
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Definition
| Sigma factor is partially displaced to allow for elongation of the transcript |
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Term
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Definition
Once ~9-12nt of RNA are synthesized, the initiation complex becomes the elongation complex
Conformational change in the core enzyme and loss of some sigma contacts
Allows for high processivity |
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Term
| Elongation complex holds open a transcription bubble in the DNA, where one NTP at a time is added to the growing RNA |
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Definition
Grows 5’ to 3’, by complementary base pairing
Strand shifts downstream one base and another NTP is added
The DNA-RNA hybrid is only ever ~9-12bp long: one nucleotide is released as another is added |
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Term
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Definition
Rho-independent termination
Rho-dependent termination |
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Term
| Rho-independent termination |
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Definition
a consensus sequence of an inverted repeat leads to a stem-loop in the transcript
This conformation causes the polymerase to stop and release the transcript |
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Term
| Rho-dependent termination |
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Definition
Requires Rho protein.
Rho can bind only where ribosomes are absent (after the stop codon)
Binds at C-rich repeat, but no consensus sequence
Rho “chases” the RNA pol, and catches it at the terminator stem-loop
Unwinds the DNA-RNA hybrid, transcription ends, all components released
*No known Rho gene in eukaryotes |
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Term
| Eukaryotes vs. Prokaryotes |
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Definition
Eukaryotes separate transcription and translation
Eukaryotes regulate gene expression more than prokaryotes |
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Term
| Eukaryotes separate transcription and translation |
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Definition
| Transcription in the nucleus, translation in the cytoplasm |
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Term
| Eukaryotes regulate gene expression more than prokaryotes |
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Definition
Much more complicated
Often different names for proteins with similar function (discovered by different people at different times) |
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Term
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Definition
Transcriptional regulatory elements upstream (5’) of a coding region constitute much of the gene length
Regulatory region of 10kb for a coding region of 2-3kb
Genes vary greatly in size: smallest only 500nt to largest 2.5 million nt (humans) |
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Term
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Definition
Gene regulatory elements are cis-acting
Trans-acting factors are transcribed and translated elsewhere and diffuse to the target, a cis-acting sequence |
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Term
| Gene regulatory elements are cis-acting |
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Definition
They are physically connected or adjacent to the coding region
This is a broad classification:
Promoter elements are close to the start of transcription, long-range regulatory elements are farther upstream |
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Term
| Trans-acting factors are transcribed and translated elsewhere and diffuse to the target, a cis-acting sequence |
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Definition
| Transcription factors are trans-acting element |
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Term
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Definition
“Gene promoter” is generally defined as: collection of cis-regulatory elements required for initiation of transcription or that increase frequency of transcription, when positioned close to the start site (+1)
Includes: core promoter and proximal promoter elements (or “upstream promoter elements” or “upstream regulatory elements”) |
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Term
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Definition
| ~60bp sequence overlapping the +1 site |
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Term
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Definition
| Lose function if moved even a short distance from +1 site or if orientation is altered |
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Term
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Definition
| General transcription factor TFIID is responsible for recognition of all core promoter elements, except BRE (uses TFIIB, B Recognition Element) |
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Term
| Proximal Promoter Elements |
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Definition
| Proximal promoter elements increase the frequency of initiation of transcription, if they are located close to the start site. |
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Term
| Proximal Promoter Elements |
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Definition
| Proximal promoter elements increase the frequency of initiation of transcription, if they are located close to the start site. |
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Term
| Proximal Promoter Elements |
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Definition
Transcription factors that bind them are not always activators or repressors.
- Sometimes serve as “tethering agent” to bring long-range regulatory elements to the core promoter |
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Term
| Proximal Promoter Elements |
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Definition
UAS – Upstream Activating Sequence (yeast)
TFIID binding to core promoter region depends on UAS
Composed of 2-3 closely linked biding sites for 1-2 different transcription factors |
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Term
| Proximal Promoter Elements |
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Definition
Higher eukaryotes have several proximal promoter elements, usually in clusters
CAAT box – bound by CBF (CAAT-binding factor), C/EBP (CAAT/enhancer-binding protein)
GC box – bound by Sp1 |
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Term
| Long-Range Regulatory Elements |
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Definition
protein coding genes contain regulatory sequences that work over large distances (100kb or more)
Enhancers and silencers
Insulators
Locus control regions (LCRs) |
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Term
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Definition
Usually 700-1000bp+ away from +1 site
Can be downstream, upstream, or within an intron
Function in same or opposite direction as promoter
Typically ~500bp in length
Contain around 10 binding sites for various transcription factors
Can be tissue- or developmental stage-specific |
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Term
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Definition
| Genome is separated into euchromatin (gene-rich) and heterochromatin (gene-poor) |
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Term
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Definition
| highly condensed and tends to spread into neighboring DNA |
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Term
| Insulators serve two functions |
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Definition
1. Chromatin boundary marker
Marks border b/w euchromatin and heterochromatin
2. Enhancer blocking activity
Keeps enhancers/silencers of one gene from acting on another |
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Term
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Definition
Typically 300-2kb in length
Contain clustered binding sites for sequence-specific DNA-binding proteins
For enhancer blocking, not certain of mechanism |
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Term
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Definition
| insulators tether DNA to subnuclear sites, forming loops, keep promoter from other enhancers |
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Term
| General Transcription Machinery |
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Definition
| 5-10% of coding capacity of genome is dedicated to proteins that regulate transcription |
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Term
General Transcription Machinery
3 major classes: |
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Definition
1. Basal transcription machinery
2. Transcription factors
3. Transcriptional coactivators/repressors |
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Term
| Basal transcription machinery |
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Definition
| components of RNA polymerase complexes |
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Term
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Definition
| sequence-specific DNA binding proteins for promoters and long-range reg. elements |
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Term
| Transcriptional coactivators/repressors |
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Definition
| increase/decrease transcription through protein-protein interactions (don’t interact with DNA) |
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Term
General Transcription Machinery
Three major components |
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Definition
1. RNA pol II – 12-subunit polymerase, synthesizes RNA and proofreads new transcript
2. General transcription factors – five of them: TFIIB, TFIID, TFIIE, TFIIF, TFIIH
Responsible for recognizing promoter and unwinding promoter DNA
RNA pol II dependent on these factors
3. Mediator – 20-subunit complex, transduces regulatory info from activator/repressor proteins to RNA pol II |
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Term
| RNA polymerase II structure |
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Definition
12 subunits – Rpb1-12
Numbered according to size, highly conserved
Catalytic core = All Rpbs but 4 and 7
C-terminal domain (CTD) |
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Term
| Catalytic core = All Rpbs but 4 and 7 |
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Definition
Still need 4 and 7 for full function
Rpb1 and 2 form central mass and positively charged “cleft” where nucleic acids bind, 9bp of DNA-RNA hybrid in center |
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Term
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Definition
Tail-like feature of largest subunit
Begins unphosphorylated for initiation, is phosphorylated for elongation and termination, dephosphorylated to begin again |
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Term
| General Transcription Factors |
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Definition
| Factors required for initiation of RNA pol II transcription at a promoter with classic TATA box and strong Inr element (in vitro) |
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Term
| General Transcription Factors |
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Definition
| Small domains of general transcription factors can enter “openings” in RNA pol II to modulate function during transcription |
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Term
| General Transcription Factors |
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Definition
| Series of highly ordered steps for general transcription apparatus assembly |
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Term
| preinitiation complex formation at promoter with TATA box |
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Definition
First, TFIID binds to TATA box (TAFs and TBP subunits interact)
Third, TFIIF and RNA pol II bind
(Mediator also binds)
Last, TFIIE and TFIIH bind downstream of RNA pol II |
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Term
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Definition
| First general transcription factor to associate with template DNA |
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Term
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Definition
| Composed of TATA box-binding factor (TBP) and 14 TBP-associate factors (TAFs) |
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Term
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Definition
| TBP has sequence-specific DNA binding at TATA box |
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Term
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Definition
TFIID binding to core promoter is rate limiting step in transcription
Activators can modulate this binding to control transcription |
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Term
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Definition
| Formation of preinitiation complex is another level for regulation |
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Term
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Definition
Helps to orient RNA pol II in the preinitiation complex
The configuration is such that DNA needs only move in a straight line to the active site.
Conservation of space from TATA box to the start site ensures +1 will be in the active center |
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Term
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Definition
Most complex general transcription factor
Phosphorylates CTD of RNA pol II
Functions in transcription and DNA repair |
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Term
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Definition
| Protein complex that acts as a bridge b/w transcriptional activators bound to enhancers and RNA pol II |
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Term
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Definition
| Not required for basal level transcription, but is needed for activator-responsive transcription |
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Term
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Definition
| Component of preinitiation complex |
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Term
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Definition
| Several Mediator subunits required for almost all genes |
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Term
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Definition
| Solely found in eukaryotes |
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Term
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Definition
Mediate gene-specific transcriptional activation/repression
Repressors block general transcription machinery
Activators increase rate of transcription 3 ways |
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Term
| Activators increase rate of transcription 3 ways |
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Definition
1. Stimulate recruitment of preinitiation complex at core promoter
2. Induce conformational change or modification (ex. Phosphorylation) to stimulate activity of general transcription machinery
3. Interact with chromatin remodeling and modification complexes to enhance access of general transcription factors to template DNA |
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