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.
RNA polymerase II core promoters are made up of combinations of ________ classes of sequence element
The eukaryotic ______ ______ refers to the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery used in vitro
A core promoter is typically ______ nucleotides long, extending either ______ or _____ from the transcription start site.
- 40-60 nucleotides
- upstream or downstream
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
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
The _____ is the most common element , found in combination with both TATA and DPEs.
RNA polymerase II forms a _______ complex with GTFs at the promoter
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
The complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation
Many pol II promoters contain a ______ element found 30bp _______ from the transcription start site.
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
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)
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
Upon binding DNA, TBP extensively ______ the TATA sequence. The resulting TBP-DNA complex provides a platform to ______ other GTFs and polymerases to the promote.
The order the proteins assemble at the promoter (in vitro)
- TFIIF with polymerase
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
Promoter escape requires __________ of the polymerase tail
Before the polymerase escapes the promoter and enters the elongation phase, there is a period of _______ initiation
the polymerase synthesizes a series of short transcripts
In eukaryotes, promoter escape involves two steps, name them
- ATP hydrolysis (for DNA melting)
- phosphorylation of the polymerase
TBP is associated with about ____ TAFs. Two of the TAFs bind DNA elements (Inr and downstream ______ elements) at the ______.
TFIIB is a protein is a single _______ _____. It enters the preinitaiton complex after _______.
TFIIF has _____ subunits in humans and associates with ______. It is recruited to the promoter with _______.
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
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.
- recruitment & regulation
- 10 polymerase
Within TFIIH are two subunits that function as ______ (involved in ______ excision repair) and another that is a ______ ______, with roles in promoter melting and escape
- nucleotide ex repair
- protein kinase
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
- flipped out
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 _____.
- double stranded
Elongating polymerase is associated with a new set of protein factors required for various types of RNA ______
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
- capping of the 5'end of the RNA, splicing and polyadenylation of the 3'end of the RNA
The most complicated of the processing events is _______. It is a process where ________ ________ are removed from RNA to generate the mature mRNA.
- noncoding introns
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?
- serine position 5
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
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
- 5'-5' linkage
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.
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
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 ____ ______.
- tail repeats
- RNA splicing
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 _________.
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
- 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
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)
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
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
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 _____.
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
Transcription termination is linked to RNA destruction by a highly ______ _____
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
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
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.
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
- 5' to 3'
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.
- conformational change
- conformational change
- CTD tail
- CTD tail
- conformational change
RNA pol I and pol III make _______ RNAs