chapter 8 part 2 Flashcards
most common eukaryotic promoter consensus sequence
TATA box located at position -25
what is another name for the TATA box?
Goldberg-Hogness box
what consensus sequence in eukaryotes is often found near the -80 position
CAAT box
what consensus sequence in eukaryotes is often found near the -90 position
GC-rich box
(5’-GGGCGG-3’)
what helps RNA pol II recognize and bind to promoter sequences?
proteins called transcription factors
what is the principle binding site for eukaryotes during promoter recognition
TATA box
where do transcription factors bind
nearby regulatory sequences and interact with RNA polymerase
TFII factors
TFs that interact with pol II
eukaryotic promoter recognition steps
- TFIID binds TATA box sequence and TBP-associated factor (initial committed complex)
- TFIIB, TFIIF, and RNA pol II join complex (minimal initiation complex)
- joined by TFIIE and TFIIH (complete initiation complex)
TFIID
multisubunit protein containing TATA-binding protein (TBP)
initial committed complex
assembled TFIID bound to TATA box
minimal initiation complex
when TFIIB, TFIIF, and RNA pol II join initial committed complex
completely initiation complex
minimal initiation complex joined by TFIIE and TFIIH
what does the complete initiation complex contain?
multiple proteins referred to as general transcription factors
Band Shift Assay
- fragments of DNA containing suspected fragments isolated
- mixed with RNA polymerase/transcription factors
- fragments that bind to RNA pol/tf have slower electrophoretic migration than unbound
is the exact location of RNA pol binding in band shift assay known?
no
DNA footprint protection assay
- first few steps indentical to band assay
- all fragments end-labeled with p32
- Dnase I added to each reaction
- all fragments subjected to electrophoresis
- gaps represent “footprint protection” by presence of bound transcriptional protein
Dnase I function
randomly cleaves DNA not protected by proteins
what do mutations inside the consensus region do
significantly reduce levels of transcription
what do mutations outside consensus region do
have non-significant effects on transcription
what do enhancer sequences do
increase level of transcription of specific genes
are enhancers DNA and RNA sequences?
DNA
where are enhancer sequence in comparison to the gene
- can be upstream or downstream
- often very different from gene sequence
what do enhancer sequences bind to
activator proteins and associated co-activators that interact with the TF attached to promoters
what do the activator/coactivator proteins form in enhancer sequences
protein bridge that links enhancer proteins to initiation complex at promoter
silencer sequences
DNA elements that act at a distance to repress transcription of target genes
what do silencers bind to
transcription factors called repressor proteins
repressor proteins
induce bends in the dNA to reduce transcription
where are silencers located
- upstream or downstream
- variable distances
where are ribosomal genes found
nucleolus
nucleolus
nuclear organelle containing rRNA and multiple copies of genes encoding rRNA
what is transcription with RNA pol I similar to
that of RNA pol II
how many functional sequences do RNA pol I promoters have near start of transcription
2
2 functional sequences of RNA pol I promoters
- core element
- upstream control element
core element
- stretches from -45 to +20
- initiation of transcription
- bound by sigma-like factor 1 (SL1) protein
upstream control element
- spans from -100 to -150
- increases transcription level
- bound by upstream binding factor 1 (UBF1)
what do most RNA pol III promoters have
internal control region
are ICRs within or outside of the transcribed region?
within the transcribed region
internal control region composition
2 short DNA sequences:
- box A and box B or C
how many base pairs separate box A and box B/C in the internal control region
25
what kind of proteins bind to the boxes in the internal control region
TFII proteins
where does transcription begin in the internal control region
near box A
what does archaeal promoter/transcription resemble?
simplified version of eukaryotic pol II
transcription promotion in archaea
- 2 proteins homologous to eukaryotic tf identify 2 promoter consensus regions
- TATA-binding protein (TBP) binds to TATA box
- TFB binds to TFB-recognition element (BRE) upstream of TATA box
- RNA pol then binds
are bacterial or eukaryotic transcripts more stable
eukaryotic
where does transcription occur in eukaryotes
nucleus
where does translation occur in eukaryotes
cytoplasm
are introns found in bacterial transcripts?
no
pre-mRNA
initial eukaryotic gene mRNA, fully processed to become mature mRNA
modifications in post-transcriptional processing
- 5’ capping
- 3’ polyadenylation
- intron splicing
when does 5’ capping start
after first 20-30 nucleotides of mRNA have been synthesized
guanylyl transferase
adds guanine to 5’ end of pre-mRNA to form unique 5’ to 5’ nucleotide bond
what do additional enzymes do during mRNA 5’ capping
methylates newly added guanine and nearby nucleotides
functions of 5’ capping
- PROTECTION of mRNA from degradation
- TRANSPORT of mRNA out of nucleus
- subsequent intron SPLICING
- enhance TRANSLATION` efficiency by orienting ribosome on mRNA
3 steps of 5’ capping
- guanylyl transferee removes y (3rd) phosphate of 5’ end of mRNA (leaves 2 phosphates)
- guanine to be added loses 2 phosphates to become monophosphate
- guanylyl transferase joins guanine monophosphate to 5’ mRNA by 5’ to 5’ triphosphate lihnkage
name of bond between phosphates in 5’ capping
5’ to 5’ triphosphate linkage
overview of 3’ polyadenylation
3’ end of pre-mRNA modified by action of several enzymes that remove a section of 3’ message and replace it with a string of adenines
functions of polyadenylation
- facilitated TRANSPORT of mature mRNA across nuclear membrane through pore complex to cytoplasm
- PROTECTS mRNA from degradation
- enhances TRANSLATION by enabling ribosomal recognition of mRNA
steps of polyadenylation
- cleavage/polyadenylation specificity factor binds near signal sequence
- cleavage-stimulating factor binds to uracil-rich region downstream
- CFI, CFII, and polyadenylate polymerase also bind
- pre-mRNA cleaved 15-30 nucleotide downstream poly. signal
- 3’ end of mRNA undergoes addition of 20-2000 A
- poly-A-binding protein joins A tail to increase rate of addition
polyadenylation signal sequence
5’ - AAUAAA - 3’
- downstream of stop codon
what happens to mRNA released by cleavage during polyadenylation
later degraded
how are 20-200 A added to 3’ end of cut pre-mRNA
through action of CPSF and PAP
when does poly-A-binding protein join adenine tail
after addition of first 10 proteins
how are polyadenylation and transcription termination connected
by activity of specialized RNase (Torpedo)
Torpedo model of transcription termination
- after 3’ cleavage/polyadenylation, uncapped transcript still attached to RNA pol
- digest residual transcript like torpedo
- RNase catches up to polymerase and triggers termination
