Term
|
Definition
| a unit of the genome in which DNA is replicated. Each contains an origin for initiation of replication |
|
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Term
|
Definition
| a sequence of DNA at which replication is initiated |
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|
Term
| An origin usually initiates... |
|
Definition
| bidirectional replication |
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|
Term
| A ______ is initiated at the origin and then moves sequentially along DNA |
|
Definition
|
|
Term
| Replication is ____ when a single replication fork is created at an origin. |
|
Definition
|
|
Term
| Replication is ____ when an origin creates two replication forks that move in opposite directions |
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Definition
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|
Term
| Each eukaryotic chromosome contains ____ replicons |
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Definition
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Term
| Because of the many replicons in eukaryotic chromosomes, the progression into ____ is tightly controlled |
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Definition
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|
Term
| Eukaryotic replicons are __ to __ kb in length |
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Definition
|
|
Term
| When are individual replicons activated during S phase? |
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Definition
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|
Term
| Regional activation patterns suggest that replicons near one another... |
|
Definition
| are activated at the same time |
|
|
Term
| Eight common characteristics between eukaryotic and prokaryotic replication |
|
Definition
-Complementary base pairing
-Semi-conservative
-Unwinds the helix and moves as forks
-Bidirectional (usually)
-Requires an RNA primer (common of all nucleotide synthesis)
-Discontinuous on lagging strand
-Discontinuous fragments referred to as Okazaki fragments
-Requires enzymes called DNA polymerases |
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|
Term
| Sites of initiation are called... |
|
Definition
|
|
Term
| In E. coli, a ____ serves as the origin for the entire bacterial chromosome and is known as... |
|
Definition
single well-defined sequence
OriC |
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Term
|
Definition
|
|
Term
| Eukaryotic origins are much more ____ than prokaryotic origins. |
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Definition
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|
Term
| Location and sequence of eukaryotic origins have been determined by ____ and ____ approaches, most notably... |
|
Definition
genetic and molecular
ARS in yeast |
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Term
| Origins in S. cerevisiae are short _-_ sequences that have an essential __ bp sequence |
|
Definition
|
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Term
|
Definition
| a complex of six proteins that binds to an ARS (origin for replication in yeast). |
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Term
| The common features among ARSes are the conserved 11 bp sequence called the... |
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Definition
|
|
Term
| Related ORC complexes are found in... |
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Definition
|
|
Term
| ____ controls eukaryotic rereplication |
|
Definition
|
|
Term
| Licensing factor is necessary for... |
|
Definition
| initiation of replication at each origin |
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|
Term
| Licensing factor is present in the ____ prior to replication, but is ____,_____, or _____ by replication |
|
Definition
nucleus
removed, inactivated, or destroyed |
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|
Term
| Each origin fires ____ per S phase. The licensing factor determines this. |
|
Definition
|
|
Term
| What is an example of a well-defined eukaryotic ori? |
|
Definition
|
|
Term
|
Definition
-confers autonomous replication to plasmids
-contains cis-acting sequences that act as origins
-SOME ARSs function as chromosomal origins
-Interacts with the ORC |
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Term
|
Definition
| Origin Replication Complex |
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Term
| Proteins of the ORC are ___-acting |
|
Definition
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|
Term
| ARS1 contains __ related 11 bp elements important for origin function. They are... |
|
Definition
|
|
Term
| Element A of ARS1 contains ____ of 11bp. |
|
Definition
| ACS (ARS Consensus Sequence) |
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|
Term
| Which element of ARS1 is absolutely critical for origin function? |
|
Definition
|
|
Term
| What is the B domain of ARS1? |
|
Definition
| a stretch of easily unwound DNA (probably A-T rich) |
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Term
|
Definition
| local unwinding at origin/"helicase activity" |
|
|
Term
| ORC is composed of _ polypeptides, they are.. |
|
Definition
6
120, 72, 62, 56, 53, and 50 kDa |
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|
Term
| What does the ORC do throughout the cell cycle and what activates it? |
|
Definition
Sits on the origin (ARS)
Activated by cdc6 |
|
|
Term
| Name the 8 players in post-ORC activation |
|
Definition
RPA (cellular replication protein A)
DNA Pol alpha/primase
RFC (replication factor C)
PCNA (proliferating cell nuclear antigen)
MF1
RNaseH
DNA ligase
DNA helicase |
|
|
Term
| What is RPA (cellular replication protein A) and what does it do? |
|
Definition
| It is a 3-subunit, ssDNA-binding protein. Also binds pol alpha |
|
|
Term
| What does DNA pol alpha/primase do? |
|
Definition
1) Synthesize 1st RNA primer, then
2) DNA on lagging strand |
|
|
Term
| What does RFC (replication factor C) do? |
|
Definition
| Binds 3' end of nascent DNA strand |
|
|
Term
| What is PCNA (proliferating cell nuclear antigen)? |
|
Definition
| a subunit of DNA pol delta |
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|
Term
| After binding 3' end, RFC loads ___ and ___ onto template, REPLACING ___ |
|
Definition
|
|
Term
| After formation, the RFC/PCNA/pol delta complex then... |
|
Definition
| extends nascent DNA strand to form the continuously synthesized leading strand complex |
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Term
|
Definition
|
|
Term
|
Definition
|
|
Term
| The ORC serves as a landmark for... |
|
Definition
|
|
Term
| ___ is an unstable protein synthesized only in G1 |
|
Definition
|
|
Term
| Cdc6 has ___ activity, so it requires energy. |
|
Definition
|
|
Term
|
Definition
|
|
Term
| Cdcc6 precedes ____ and... |
|
Definition
| MCMs and allows them to bind |
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Term
|
Definition
| mini chromosome maintenance |
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|
Term
| Licensing factor includes... |
|
Definition
-Cdc6 (destroyed, must be re-synthesized) -> ~half life: <5 minutes
-MCM 2,3,5
-possibly others
-CdtI likely tethers |
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|
Term
| When replication is initiated, ___,___, and ___ proteins are displaced. |
|
Definition
|
|
Term
| The degradation of ___ prevents reinitiation, thanks to... |
|
Definition
|
|
Term
|
Definition
| A protein-DNA complex at the origin in S. cerevisiae that is required for DNA replication. |
|
|
Term
| What does the prereplication complex contain? |
|
Definition
| ORC complex, Cdc6, MCM proteins |
|
|
Term
|
Definition
| A protein-DNA complex in S. cerevisiae that consists of the ORC complex bound to the origin |
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|
Term
| Some MCM proteins are in the ____ throughout the cell cycle, but others may enter only after ____ |
|
Definition
|
|
Term
| Nascent DNA strands synthesized by semi-conservative replication have... |
|
Definition
| different modes of synthesis |
|
|
Term
| The DNA polymerase ____ when it synthesizes the leading strand, but synthesizes the lagging strand by... |
|
Definition
advances continuously
making short fragmnets (Okazaki fragments) that are subsequently joined together |
|
|
Term
| semidiscontinuous replication |
|
Definition
| the mode of replication in which one new strand is synthesized continuously while the other is synthesized discontinuously |
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|
Term
| ____ enzyme units are required to synthesize the leading and lagging strands |
|
Definition
|
|
Term
| ____ eukaryotic DNA polymerases undertake initiation and elongation |
|
Definition
|
|
Term
| A replication fork has one complex of ____ and two complexes of _____ |
|
Definition
DNA polymerase alpha/primase
DNA polymerase delta and/or epsilon |
|
|
Term
| The DNA polymerase alpha/primase complex initiates... |
|
Definition
| the synthesis of both DNA strands |
|
|
Term
| DNA polymerase epsilon... |
|
Definition
| elongates the leading strand and a second DNA polymerase delta elongates the lagging strand |
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|
Term
| Homologous recombination can occur... |
|
Definition
| at any point along the lengths of two homologous chromosomes |
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Term
|
Definition
| essential in meiosis for generating diversity and for chromosome segregation, and in mitosis to repair DNA damage and stalled replication forks |
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|
Term
| Site-specific recombination occurs between... |
|
Definition
| two specific sequences. The other sequences in the two recombining DNAs are not homologous |
|
|
Term
| Homologous recombination occurs between... |
|
Definition
| synapsed chromosomes in meiosis |
|
|
Term
|
Definition
| each of two identical copies of a replicated chromosome; this term is used as long as the two copies remain linked at the centromere |
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|
Term
| Sister chromatids separate during... |
|
Definition
| anaphase in mitosis or anaphase II in meiosis |
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|
Term
|
Definition
| the structure containing all four chromatids (two representing each homolog) at the start of meiosis |
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|
Term
| Recombination involves pairing between... |
|
Definition
| complementary strands of the two parental DNAs |
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|
Term
| Chromosomes must ___ in order for chiasmata to form where crossing over occurs |
|
Definition
|
|
Term
| Stages of meiosis are correlated with... |
|
Definition
| the molecular events that occur to DNA |
|
|
Term
|
Definition
| the protein structure that forms between synapsed homologous chromosomes that is believed to be necessary for recombination to occur |
|
|
Term
|
Definition
| a pair of DNA duplexes that are connected together through a reciprocal exchange of genetic material |
|
|
Term
| Each pair of sister chromatids has an axis made of... |
|
Definition
|
|
Term
| ____ and ____ form the lateral elements and transverse filaments/central elements |
|
Definition
| cohesins and zip proteins |
|
|
Term
|
Definition
| a proteinaceous structure around which the chromosomes condense at the start of synapsis |
|
|
Term
|
Definition
| a structure in the synaptonemal complex that forms when a pair of sister chromatids condenses onto an axial element |
|
|
Term
| Molecular basis of crossing over |
|
Definition
-Pairing of homologous non-sister chromatids and formation of synaptonemal complex
-Exact lining up of DNA duplex
-Endonuclease nick in single strand
-Denaturation or separation of strands
-Reassociation of complete strands
-Broken ends sealed by a ligase
-Holliday intermediate formed
-Strands opposite crossover strands are nicked by endonuclease
-Digestion of single strand pieces by nuclease
-Rotation of axis
-New DNA synthesis 5'->3'
-Gaps filled by polymerase and sealed by ligase action
Breakage, Reunion, Reciprocal exchange |
|
|
Term
| Double-strand breaks initiate... |
|
Definition
|
|
Term
|
Definition
| replaces any material that has been degraded |
|
|
Term
| In a recombinant joint molecule, the two DNA duplexes are connected by... |
|
Definition
| heteroduplex DNA and two Holliday junctions |
|
|
Term
|
Definition
| relax or introduce supercoils in DNA |
|
|
Term
| Topoisomerases alter supercoiling by |
|
Definition
| breaking bonds in DNA, changing the conformation of the double helix in space and remaking the bonds |
|
|
Term
|
Definition
| breaking a single strand of DNA |
|
|
Term
| Type II enzymes act by... |
|
Definition
| making double-strand breaks |
|
|
Term
| Example of Robertsonian translocation |
|
Definition
| Two acrocentrics fuse to form a single metacentric. Opposite event is called dissociation |
|
|
Term
| RNA sequence is ____ to template strand and ____ to coding strand |
|
Definition
|
|
Term
| The function of RNA polymerase is to... |
|
Definition
| copy one strand of the duplex DNA into RNA |
|
|
Term
| A transcription unit is... |
|
Definition
| a sequence of DNA transcribed into a single RNA, starting at the promoter and ending at the terminator |
|
|
Term
| 3 stages of transcription + descriptions |
|
Definition
-Initiation: Template recognition-RNA polymerase binds to duplex DNA, DNA is unwound at promoter, very short chains are synthesized and released
-Elongation: polymerase synthesizes RNA
-Termination: RNA polymerase and RNA are released |
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|
Term
|
Definition
|
|
Term
|
Definition
| destabilizes non-specific binding |
|
|
Term
|
Definition
|
|
Term
| The holoenzyme is made up of... |
|
Definition
|
|
Term
| Explain the three mechanisms for how RNA polymerase finds a promoter |
|
Definition
-Sliding: exactly what it sounds like
-Intersegment transport: Jumps to another segment above/below
-Intrasegment transfer/intradomain association and dissociation or hopping: Hops along strand |
|
|
Term
| The rate at which RNA polymerase binds to promoters can be... |
|
Definition
| too fast to be accounted for by random diffusion |
|
|
Term
| RNA polymerase probably binds to... |
|
Definition
| random sites on DNA and exchanges them with other sequences very rapidly until a promoter is found |
|
|
Term
|
Definition
| -55 to +20, contains sigma and covers ~75 bp |
|
|
Term
| Initial elongation complex |
|
Definition
| forms at 10 bases, may lose sigma, and loses contacts from -35 to -55 |
|
|
Term
| General elongation complex |
|
Definition
| forms at 15-20 bases and covers 30-40 bp |
|
|
Term
| Sigma factor and core enzyme recycle at... |
|
Definition
| different points in transcription |
|
|
Term
|
Definition
|
|
Term
| How many promoters does Sigma 70 recognize? |
|
Definition
|
|
Term
| E. coli sigma factors recognize promoters with... |
|
Definition
| different consensus sequences |
|
|
Term
| Promoter recognition depends on... |
|
Definition
|
|
Term
| The promoter consensus sequences consist of... |
|
Definition
a purine at the startpoint
the hexamer TATAAT centered at -10
another hexamer centered at -35 (-35 box) |
|
|
Term
| Individual promoters usually... |
|
Definition
| differ from the consensus at one or more positions |
|
|
Term
| Promoter efficiency can be affected by... |
|
Definition
| additional elements as well |
|
|
Term
|
Definition
| decrease promoter efficiency and decrease conformance to the consensus sequences |
|
|
Term
|
Definition
| increase promoter efficiency and increase conformance to the consensus sequences |
|
|
Term
| Mutations in the ___ sequence... |
|
Definition
| can affect initial binding of RNA polymerase |
|
|
Term
| Mutations in the __ sequence... |
|
Definition
| usually affect the melting reaction that converts a closed to an open complex |
|
|
Term
| What amino acids of the 2.4 helix of sigma 70 contact specific bases in the coding strand of the -10 promoter element? |
|
Definition
|
|
Term
| The ____ of sigma blocks the DNA-binding regions from binding to DNA |
|
Definition
|
|
Term
| How does the N-terminus of sigma block DNA binding? |
|
Definition
| N-terminal region binds DNA-binding domains in free sigma. DNA then displaces N-terminus when complex forms with DNA |
|
|
Term
| Sigma 70 changes its structure to... |
|
Definition
| expose its DNA binding regions when it associates with the core enzyme |
|
|
Term
| Sigma 70 finds both the ___ and ___ sequences |
|
Definition
|
|
Term
| The DNA sequences required for termination are located _____ of the terminator sequence. Formation of a _____ in the RNA may be necessary. |
|
Definition
|
|
Term
| Intrinsic termination requires... |
|
Definition
| a hairpin and U-rich region |
|
|
Term
| Intrinsic terminators consist of a... |
|
Definition
| G-C-rich hairpin in the RNA product followed by a U-rich region in which termination occurs |
|
|
Term
| Intrinsic terminators include ____ regions that can form hairpins varying in length from __ to __ bp |
|
Definition
|
|
Term
| What does the Rho factor do? |
|
Definition
| binds to RNa at a rut site and translocates along RNA until it reaches the RNA-DNA hybrid in RNA polymerase, where it releases the RNA from the DNA |
|
|
Term
| Describe the steps of Rho's interaction with RNA polymerase |
|
Definition
-RNA polymerase transcribes DNA
-Rho attaches to rut site on RNA
-Rho translocates along RNA
-RNA pol pauses at hairpin and rho catches up
-Rho unwinds DNA-RNA hybrid
-Termination: all components released |
|
|
Term
|
Definition
| a terminator protein that binds to a rut site on a nascent RNA and tracks along the RNA to release it from the RNA-DNA hybrid structure at the RNA polymerase |
|
|
Term
|
Definition
| Rho Utilization Site, the sequence of RNA recognized by the rho termination factor |
|
|
Term
| What are the % of bases in a rut site? |
|
Definition
|
|
Term
| What are the two domains of the Rho monomer? |
|
Definition
N-terminal RNA binding domain
C-terminal ATPase domain |
|
|
Term
| How many monomers make up Rho? |
|
Definition
|
|
Term
| The sigma factor associated with core polymerase determines... |
|
Definition
| the set of promoters at which transcription is initiated |
|
|
Term
| Substitution of sigma factors may control... |
|
Definition
|
|
Term
| How many sigma factors does E coli have? |
|
Definition
|
|
Term
| Sigma __ is used for general transcription |
|
Definition
|
|
Term
| A ____ is created when one sigma factor is required to transcribe the gene coding for the next sigma factor |
|
Definition
|
|
Term
| What do antiterminator proteins do? |
|
Definition
| enable RNA polymerase to pass terminator |
|
|
Term
| What antitermination protein is used for immediate early transcription units? |
|
Definition
|
|
Term
| What antitermination protein is used for late transcription units? |
|
Definition
|
|
Term
| Transcription and translation occur ____ in bacteria |
|
Definition
|
|
Term
| couple transcription/translation |
|
Definition
| as ribosomes begin translating an mRNA before its synthesis has been completed |
|
|
Term
| How stable is bacterial mRNA? |
|
Definition
| Not very---half life of only a few minutes |
|
|
Term
|
Definition
| an RNA chain that is still being synthesized, so that its 3' end is paired with DNA where RNA polymerase is elongating |
|
|
Term
|
Definition
| mRNA that encodes one protein |
|
|
Term
| A bacterial mRNA may be polycistronic in.. |
|
Definition
| having several coding regions that represent different cistrons |
|
|
Term
|
Definition
| the untranslated sequence upstream from the coding region of an mRNA |
|
|
Term
|
Definition
| the untranslated sequence downstream from the coding region of an mRNA |
|
|
Term
|
Definition
| in polycistronic mRNA, the disgance between the termination codon of one cistron and the initiation codon of the next cistron |
|
|
Term
| Two examples of in vivo eukaryotic transcription assay systems |
|
Definition
1) Xenopus oocytes and embryos
2) Transfected mammalian cells |
|
|
Term
| One advantage of transient in vivo transfected mammalian cells |
|
Definition
| the promoters are not affected by the position effect |
|
|
Term
|
Definition
| Cells transfected with DNA constructs that integrate into the genome. Integration can involve recombination or the use of retroviral constructs. This allows long term expression studies. |
|
|
Term
|
Definition
| transfected with non-integrating plasmid. Expression can be followed for up to 3-4 days. Plasmids expressing factors often utilize viral promoters that are very strong |
|
|
Term
| Chromatin must be ____ before RNA polymerase can bind the promoter |
|
Definition
|
|
Term
| basal transcription factors |
|
Definition
| TFs required by the RNA polymerase to form the initiation complex at all promoters |
|
|
Term
|
Definition
| TF_N_X, where N is I, II, or III and X is a letter |
|
|
Term
| RNA polymerase I synthesizes... |
|
Definition
|
|
Term
| RNA polymerase II synthesizes... |
|
Definition
|
|
Term
| heterogeneous nuclear RNA (hnRNA) |
|
Definition
| RNA that comprises transcripts of nuclear genes made primarily by RNA polymerase II; it has a wide size distribution and variable stability |
|
|
Term
| RNA polymerase III synthesizes... |
|
Definition
| small RNAs in the nucleoplasm |
|
|
Term
| All eukaryotic RNA polymerases have... |
|
Definition
| ~12 subunits and are aggregates of ~500 kD |
|
|
Term
|
Definition
| common to all 3 RNA polymerases |
|
|
Term
| The largest subunit in RNA polymerase II has a... |
|
Definition
| CTD (carboxy-terminal domain) consisting of multiple repeats of a heptamer |
|
|
Term
| For most biologists, transcriptional studies can be split into two types... |
|
Definition
1) Promoter analysis
2) Basic mechanisms |
|
|
Term
|
Definition
| Where and when? What DNA sequences are important in controlling the spatial/temporal/level of transcriptional activity |
|
|
Term
|
Definition
|
|
Term
|
Definition
| Upstream Activation Sequence |
|
|
Term
|
Definition
| the shortest sequence at which an RNA polymerase can initiate transcription (typically at a much lower level than that displayed by a promoter containing additional elements) |
|
|
Term
| What is included in the core promoter for RNA polymerase II |
|
Definition
the minimal sequence at which the basal transcription apparatus can assemble, often includes one or more of three sequence elements: the Inr, the TATA box, and the DPE
Typically ~40 bp long |
|
|
Term
|
Definition
| a cis-acting sequence that increases the utilization of most eukaryotic promoters and can function in either orientation and in any location relative to the promoter |
|
|
Term
|
Definition
| a short sequence of DNA that can inactivate expression of a gene in its vicinity |
|
|
Term
| What are the 3 most common techniques used to identify important sequences within a promoter? |
|
Definition
1) Deletion mapping
2) Linker scanning
3) DNA footprinting |
|
|
Term
|
Definition
Tx factors and activators are proteins that possess a generalized structure consisting of at LEAST three largely (and often) independently acting domains
DNA binding domain
Dimerization domain
Activation (protein-protein) domain |
|
|
Term
| Name 3 DNA binding domains |
|
Definition
Zinc finger
Homeodomain
POU domain |
|
|
Term
| Basic leucine zipper (b-zip) |
|
Definition
| Basic region contacts the DNA while the leu zipper acts as the dimerization domain. Both are required for the functional dimer to be formed and bind DNA |
|
|
Term
|
Definition
| transcription factors that are activated by binding of a steroid ligand |
|
|
Term
|
Definition
| the motif that describes an arrangement of two a-helices that form a site that binds to DNA, one fitting into the major groove and the other lying across it |
|
|
Term
| Amphipathic helices promote ____ while a nearby basic region contacts the DNA |
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
| a DNA binding motif that typifies a class of transcription factors that contain the helix-tun-helix structure that typifies a class of transcription factors often found in developmentally regulated genes |
|
|
Term
|
Definition
| the motif responsible for the dimerization of a class of transcription factors called HLH proteins |
|
|
Term
| RNA polymerase II requires general transcription factors called ____ to initiate transcription |
|
Definition
|
|
Term
| RNA polymerase II promoters frequently have the short conserved sequence _____ at the start point |
|
Definition
|
|
Term
| The TATA box is a common component of RNA polymerase II promoters and consists of... |
|
Definition
| an A-T-rich octamer located ~25 bp upstream of the start point |
|
|
Term
| downstream promoter element (DPE) |
|
Definition
| common component of RNA pol II promoters that DO NOT CONTAIN A TATA BOX |
|
|
Term
| A core promoter for RNA polymerase II includes the __ and either a __ or __ |
|
Definition
|
|
Term
|
Definition
| a component of the positioning factor that is required for each type of RNA polymerase to bind its promoter |
|
|
Term
| The factor for RNA pol II is ___ which consists of.. |
|
Definition
TF2D
TBP and ~11 TAFs with a total mass ~800 kD |
|
|
Term
|
Definition
| the TATA box in the minor groove of DNA |
|
|
Term
| TBP forms a saddle around DNA and bends it by... |
|
Definition
|
|
