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Transcription in eukaryotes is undertaken by polymerses closely related to the polymerases found in prokaryotes. However, there are differences in the machinery used in each case name two
- bacteria have only one RNA polymerase but eukaryotes have at least three different ones (pol I, II, III) **(pol IV and pol V in plants)
- bacteria require only one additional initiation factor (σ), but eukaryotes require several initiation factors (general transcription factors aka GTFs) for efficient, promoter specific initiaton.
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RNA polymerase II core promoters are made up of combinations of ________ classes of sequence element
different
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The eukaryotic ______ ______ refers to the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery used in vitro
core promoter
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A core promoter is typically ______ nucleotides long, extending either ______ or _____ from the transcription start site.
- 40-60 nucleotides
- upstream or downstream
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Elements found in Pol II core promoters (relative to the transcription start site) (4)
- TFIIB recognition element (BRE)
- The TATA element (or box)
- The initiator (Inr)
- The downstream promoter elements: DPE, DCE, and MTE
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Typically, a promoter includes some subset of those four elements. State an example
promoters typically having either a TATA element or a DPE element, but not both
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The _____ is the most common element , found in combination with both TATA and DPEs.
Inr (initiator)
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RNA polymerase II forms a _______ complex with GTFs at the promoter
preinitiation
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The ______ collectively perform the functions perform function of σ in bacterial transcription. So they help polymerase bind to the _____ and ______ the DNA. They also help polymerase ______ the promoter and embark on the _______ phase
- GTFs
- promoter
- melt
- escape
- elongation
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preinitation complex
The complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation
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Many pol II promoters contain a ______ element found 30bp _______ from the transcription start site.
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The TATA element is recognized by the GTF called ______. The nomenclature _____ denotes a transcription factor for Pol II. Like many of the GTFs, it is a _____ _____ complex
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The component of TFIID that binds to the TATA DNA sequence is called ______ _____ ______. The other subunits in this complex are called ______ ______ ______.
- TATA binding proteins (TBPs)
- TBP association factors (TAFs)
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Some TAFs recognize other core promoter elements such as _____, ______ and _____ although the strongest binding is between _____ and _______. Thus TFIID is a critical factor in _______ recognition and _______ complex establishment
- Inr, DPE, and DCE
- TBP and TATA
- promoter
- preinitiation
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Upon binding DNA, TBP extensively ______ the TATA sequence. The resulting TBP-DNA complex provides a platform to ______ other GTFs and polymerases to the promote.
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The order the proteins assemble at the promoter (in vitro)
- TFIIA
- TFIIB
- TFIIF with polymerase
- TFIIE
- TFIIH
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Formation of the preintiation complex containing these components is followed by ______ _______. In contrast to bacteria, in eukaryotes this action requires ____ _______ and is mediated by ______
- promoter melting
- ATP hydrolysis
- TFIIH
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Promoter escape requires __________ of the polymerase tail
phosphorylation
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Before the polymerase escapes the promoter and enters the elongation phase, there is a period of _______ initiation
abortive
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abortive initiation
the polymerase synthesizes a series of short transcripts
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In eukaryotes, promoter escape involves two steps, name them
- ATP hydrolysis (for DNA melting)
- phosphorylation of the polymerase
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TBP is associated with about ____ TAFs. Two of the TAFs bind DNA elements (Inr and downstream ______ elements) at the ______.
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TFIIB is a protein is a single _______ _____. It enters the preinitaiton complex after _______.
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TFIIF has _____ subunits in humans and associates with ______. It is recruited to the promoter with _______.
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Binding of ______ to ______stabilizes the DNA-TBP-TFIIB complex and is required before _____ and _____ are recruited to the preinitiation complex
- Pol II to TFIIF
- TFIIE and TFIIH
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TFIIE consists of _____ subunits, binds after ______. It has roles in ______ and ______ of TFIIH. TFIIH controls the ____ dependent transition of the preinitiation complex to the open complex. TFIIH is also the _______ and ______ complicated of the GTFs. It has _____ subunits and a molecular mass comparable to the ______ itself.
- two
- TFIIF
- recruitment & regulation
- ATP
- largest
- most
- 10 polymerase
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Within TFIIH are two subunits that function as ______ (involved in ______ excision repair) and another that is a ______ ______, with roles in promoter melting and escape
- ATPase
- nucleotide ex repair
- protein kinase
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In bacteria, melting of the 10 element promoter is mediated by bases on the _______ DNA strand being _______ out and bound within pockets in the σ subunit. This ______ (does/doesn't) require ATP hydryolysis and is driven simply by _____ reactions that favor the _______ conformation
- noncoding
- flipped out
- doesn't
- binding
- melted
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In eukaryotes the TFIIH acts as an ATP-driven _______ of double stranded DNA. This subunit binds to DNA _____ from the polymerase and feeds _____ _____ DNA with right handed threading, into the ______ of the polymerase. This action drives the _____ of the DNA because the upstream promoter DNA is held in a ______ position by ______ and the rest of the _____.
- translocator
- downstream
- double stranded
- cleft
- melting
- TFIID
- GTFs
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Elongating polymerase is associated with a new set of protein factors required for various types of RNA ______
processing
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Once transcribed, eukaryotic RNA has to be processed in various ways before being _______ from the nucleus where it can be _______. Name the three processing events
- exported
- tranlated
- capping of the 5'end of the RNA, splicing and polyadenylation of the 3'end of the RNA
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The most complicated of the processing events is _______. It is a process where ________ ________ are removed from RNA to generate the mature mRNA.
- splicing
- noncoding introns
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There is an overlap in _______ involved in elongation and those required for RNA processing. In one 5 also helps to case, an elongation factor SPT5 also helps to recruit the ______ enzyme to the CTD tail of polymerase. Where is it phosphorylated?
- proteins
- 5'capping
- serine position 5
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In another case, elongation factor TAT-SF1 recruits components of the splicing machinery to polymerase with with a Ser-2 ________ tail. Thus _______, _______ of transcription and RNA _______ are interconnected, to ensure their proper coordination
- phosphorylated tail
- elongation
- termination
- processing
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The first RNA processing event is _______. This involves the addition of a modified _______ base to the to the _____ of the RNA. Specifically, the base added is ______ ______, and it is joined to the RNA transcript by an unusual ______ linkage involving _____ phosphates
- capping
- guanine
- 5'end
- methylguanine
- 5'-5' linkage
- three
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The 5' cap is created in three enzymatic steps (mini story: 3)
- First, a phosphate group is removed from the 5'end of the transcript
- Second, the GMP moiety is added
- Finally, that nucleotide is modified by the addition of a methyl group.
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The RNA is capped as soon as it emerges from the _____ _____ _______ of polymerase. This happens when the transcription cycle has progressed only as far as the transition from the ______ to _____ phases.
- RNA exit channel
- initiation to elongation
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After capping, _________ of Ser-5 within the tail repeats may be responsible for dissociation of the capping machinery, and further ________ (of Ser-2 within the ____ _____) causes recruitment of the machinery needed ____ ______.
- dephosphorylation
- phosphorylation
- tail repeats
- RNA splicing
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The final RNA processing event is _________ of the 3' end of the mRNA . It is intimately linked with the _______ of transcription. Just as with capping splicing, the polymerase CTD tail is involved in recruiting some of the enzymes necessary for _________.
- polyadenylation
- termination
- polyadenylation
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Once polymerase has reached the end of the a gene, it encounters specific _______ that, after being ________ into RNA, trigger the transfer of ________ enzymes to that RNA, leading to four events. Name them
- sequences
- transcribed
- polyadenylation
- First, addition of many adenine residues to its 3' end
- Second, degradation of the RNA remaining associated with RNA polymerase by a 5' to 3' ribonculease
- Third, termination
- Fourth, transcription
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Which two protein complexes are carried by the CTD of polymerase as it approaches the end of the gene
- CPSF (cleavage and polyadenylation specificity factor)
- CSTF (cleavage stimulation factor)
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The sequences that, once transcribed into RNA, trigger transfer of CPSF and CSTF to the RNA are called _____ _______. Once CPSF and CSTF are bound to the RNA, other proteins are recruited as well, leading initially to RNA _______ and then ________.
- poly-A signals
- cleavage and then polyadenylation
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Polyadenylation is mediated by an enzyme called _____ ________, and it adds approx. _____ adenines to the RNA's ______ produced by the cleavage.
- poly-A polymerase
- 200 adenines
- 3'end
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Poly-A polymerase uses _____ as a precursor and adds nucleotides using the same chemistry as RNA polymerase. However, it does so without a _______. Thus, the long tail of As is found in the _____ but not in the _____.
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What terminates transcription by polymerase? **
In fact, the enzyme does not terminate immediately after the RNA is cleaved and polyadenylated. Rather, it continues to move along the template, generating a second RNA molecule. The polymerase can continue transcribing for several thousand nucleotides before terminating and dissociating from the template
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Transcription termination is linked to RNA destruction by a highly ______ _____
processive RNase
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Polyadenylation is linked to termination, although it is unclear how. An enzyme that ______ the second RNA as it emerges from the polymerase has been identified, and this enzyme may itself trigger _______. This is called the _______ model
- degrades
- termination
- torpedo
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According to the torpedo model, the free end of the second RNA is ______ and thus can be distinguished from genuine transcripts. This new RNA is recognized by an ______ called
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The RNase in the torpedo model is called _____ in yeasts and _____ in humans. It is loaded onto the end of the RNA by another protein (Rtt103) that binds the _____ of RNA polymerase.
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Rat1 enzyme is very _______ and quickly degrades the RNA in a _______ direction, until it catches up to the ________ polymerase from which the RNA is being spewed. Termination may not require any specific interaction between ____ and the polymerase. It may be triggered in a manner rather similar to _____ _______ termination in bacteria
- processive
- 5' to 3'
- transcribing
- Rat1
- Rho-dependent
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Although the torpedo model for termination is now the favored one, there is an alternative called the _______ model. According to this model, termination depends on a _________ change in the elongating polymerase that reduces the ________ of the enzyme leading to spontaneous termination soon afterward. The _________ change would be linked to polyadenylation and could, for example, be triggered by the transfer of 3'-processing enzymes from the _____ _____ of polymerase to the RNA or by binding of the ____ _____ of other factors that induce a _______ change.
- allosteric
- conformational change
- processivity
- conformational change
- CTD tail
- CTD tail
- conformational change
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RNA pol I and pol III make _______ RNAs
small
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