chapter 8 part 2 Flashcards
Where is the principal binding site for transcription factors in most eukaryotic promoters?
the TATA box
where does transcription in eukaryotes take place?
the nucleus
Which structure, located in the promoter, facilitates the binding of RNA polymerase III to tRNA promoters?
internal control region (ICR)
what enzyme permits a 5’ capping of mRNA?
guanylyl transferase
Which DNA regulatory sequence interacts with activator proteins to increase the level of transcription?
enhancer
Which of the following research techniques would be used to verify that a DNA sequence of interest binds to a transcription factor?
band shift assay
what is the most common eukaryotic consensus sequence?
the TATA Box
what is the TATA box also called?
the Goldberg-Hogness box
where is the TATA Box located?
about the -25 position
whare is the CAAT box found?
-80 position
where is the GC-rich box found?
the -90 position
do prokaryotic promoters display more or less variation than eukaryotic promoters?
less
what helps RNA pol II bind to the promoter sequence?
transcription factos (TFs)
what is the principle binding site during promoter recognition?
the TATA Box
what do TFs bind to and what do they do?
nearby regulatory sequences and they interact with RNA polymerase
what are the TFs called that interact with Pol II?
TFII Factors
what does TFIID contain?
the TATA-Binding protein
what does the TFIID bind?
it binds the TATA Box sequence and the TBP-associated factor (TAF)
what froms the initial committed complex?
the TFIID bound to the TATA Box
what makes up the minimal initation complex?
the TFIIB, TFTTF, and RNA Pol ii
what form the complete initation complex?
TAFIIE and TFIIH
what are the proteins in the complete initiation complex referred to as?
the General Transcription Factors (GTFs)
what happens in Band Shift Assay?
-isolate fragements of DNA containing suspected DNA fragments
-DNA fragments r mixed with RNA pol and/or transcription factors
-the fragments that bind to the RNA/ TFS have slower electrophoretic migration that the unbound sequences
-inidcated that the DNA fragments r bound by transcriptional proteins
is exact location known in Band Shift Assay?
No- we just know that the DNA is bound by transcriptional proteins
what happens in DNA Footprint Protection Assay?
-first couple of steps are identical to band assay
-the fragments are labeled with 32P
-Dnase I is added to each reaction (DNA not protected by proteins is cleaved)
-All fragments from both reactions are subjected to electrophoresis
is exact location known in DNA Footprint Protection Assay?
Yes- we get to know where within a fragment a transcriptional protein binds
what does Dnase I do?
it randomly gets rid of DNA not protected by proteins
Mutations inside the consensus region significantly _________ levels of transcription
reduce
Mutations outside the consensus region have ________________ effects on transcription
non-significant
what do enhancer sequences do?
Increase the level of transcription of specific genes
what do enhancer sequences bind?
activator proteins and co-activators that form a protein bridge
what does the protein bridge in enhancers link?
links to proteins at the enhancer sequence to the initiation complex at the promoter
why does the bridge bend the DNA?
so the enhancer sequence part and the initiation complex’s proteins are close enough to interact
what are silencer sequences
-reverse of enhancer sequences
-DNA elements that act at a distance to REPRESS transcription of their target genes
what do silencers bind?
transcription factors called repressor proteins that induce bend in DNA
what do the bends do in the silencers?
it reduced transcription of the target gene
where would silencer be loated?
upstream or downstream from their target gene- the distance varies
what does RNA pol I do?
transcribes genes for rRNA
where are ribosomal genes found?
in the nucleolus
what is the nucleolus?
a nuclear organelle containing r RNA and multiple copies of genes encoding rRNA
how many function sequences do RNA pol I promoters have?
2 (near the start of transcription)
what are the two functional sequences that RNA Pol I Promoters have?
- the core element
- upstream control element
core element
t (-24 to +20, needed for initiation of transcription, bound by sigma-like factor 1 (SL1) protein
upstream control element
-100 to -150, increases level of transcription, bound by upstream binding factor 1 (UBF1)
what is RNA Pol iii primarily responsible for transcription of?
tRNA genes, also does one rRNA, and other RNA-encoding genes
what do most RNA pol ii promoters have?
an internal control region (ICR)
what are ICRs
they are within the transcribes region
-typically contain dna sequences called box A and box B, or box A and box C
-25 bp seperate the two box regions
-TFII proteins bind to the two box region
-RNA pol iii n=binds to the complex and transcription begins near box A after proper RNA pol iii positioning
how many proteins identify the two promoter consensus regions in archaea
two
what is archaea transcription kind of similar too, just simplifed?
eukaryotic transcription
how does transcription work in archaea
TATA-binding protein (TBP) binds to the TATA box and transcription factor B (TFB) binds to a TFB-recognition element (BRE) upstream do the TATA box. rna pol then binds
____________ transcripts are more ________ than bacterial
transcripts
Eukaryotic
stable
what are transcription and translation seperated in time and location in?
eukaryotes
where does transcription occur in eukaryotes?
nucleus
where does translation occur in eukaryotes?
cytoplasm
what do eukaryotic transcripts have than bacterial don’t?
introns
what is the initial eukaryotic gene mRNA called?
pre-mRNA
what is the fully processed eukaryotic gene mRNA called?
mature mRNA
what do mature mRNA modifications include?
- 5’ capping
- 3’ polyadenylation
- intron splicing
when does guanylyl transferase come in to play?
after the first 20 to 30 nucleotides of mRNA have been synethesized
what does guanylyl transferase do?
it adds a guanine to the 5’ end of the pre-MRNA - unique to a 5’ to 5’ nucleotide bond
what are the several functions that the 5’ capping serves?
-protection of mRNA from degredation
-transport of mRNA out the nucleus
-intron splicing
-enhancing translation by efficiently orienting the ribosome on the mRNA
what phosphate does guanylyl transferase remove?
the y (or 3’ phosphate) of the 5’ end of the mRNA, this leaves us two phosphates
how many phosphates does the guanine to be added to the mRNA lose to become guanine monophosphate?
two
what does guanylyl transferase join the guanine monophosphate by to the 5’ mRNA by?
a 5’ to 5’ triphosphate linkage
what is pre-mRNA 3’ polyadenylation?
when the 3’ end of the pre-mRNA is modified by the action of several enzymes that remove a section of the 3’ message and replace it with a string of adenines
what are the functions of polyadenylation?
-facilitate transport of mature mRNA across the nuclear membrane to cytoplasm
-protecting mRNA from degradation
-enhancing translation by enabling the ribosomal recognition of mRNA
what type of eukaryotic transcripts dont undergo polyadenylation?
histone genes
what are the steps of polyadenylation?
- cleavage and polyadenylation specificity factor (CPSF) binds near the polyadenylation signal sequence, downstream of the stop codon
-followed by binding of cleavage stimulating factor (CstF) to a uracil rich region downstream of the polyadenylation signal sequence
-. two other cleavage factors (CFI and CFII) and polyadenylate polymerase (PAP) also bind - the pre-mrna is cleaved 15-30 nucleotides downstream of the polyadenylation signal sequence
- the 3’ end pf the cut-premRNA undergoes enzymatic addition of 20 to 200 adenines through the action of CPSF and PAP
- after addition of the first 10 adenines, molecules of poly-A-binding protein (PABII) join the adenine tail and increase the rate of addition of adenines
what is the polyadenylation signal sequence?
5’ - AAUAAA - 3’
what is the torpedo model of transcription?
it happens after the 3’ cleavage and polyadenylation when the resulting residual transcript is still attached to RNA pol ii
_____________ and transcription termination are connected by the activity of a specialized ______.
polyadenylation, RNase
what does the specialized RNase do?
it rapidly digests the residual transcript like a “torpedo” aimed at the residual mRNA attached to RNA pol II
what does RNase do when it catches up to RNA pol II
it triggers termination with RNA pol II releasing from the DNA
