Transcription Flashcards
1
Q
What enzyme synthesizes RNA?
A
RNA polymerases
2
Q
What direction is new RNA synthesized?
A
5’ to 3’
3
Q
Is a primer needed to initiate RNA synthesis?
A
NO primer is needed
4
Q
Does RNA polymerases proofread?
A
NO (there are several mRNA copies so an occasional error is OK
5
Q
What is required for synthesis of RNA?
A
a DNA template, (ribo)nucleotide triphosphate and Mg2+ (a cofactor)
6
Q
What does tRNA do?
A
brings each individual amino acid to the growing protein chain
7
Q
What is a haloenzyme?
A
a core enzyme + a sigma factor
8
Q
What is a sigma factor?
A
a sixth subunit required for efficient initiation
9
Q
What does Ecoli use for all transcript synthesis?
A
a SINGLE RNA polymerase for rRNA, tRNA, and mRNA
10
Q
What type of enzyme is RNA polymerase ?
A
a multisubunit enzyme
11
Q
What does a core enzyme contain?
A
5 subunits: 2 alpha subunits and 1 each of beta, beta prime, and omega
12
Q
What are core enzyme very INEFFICIENT at?
A
at initiating RNA synthesis
13
Q
What are the 3 stages of RNA synthesis?
A
initiation
elongation
termination
14
Q
Where is transcription initiated?
A
at specific sites (promoters) with respect to the template/coding strand
15
Q
Where are promoters found?
A
at the 5’ end of the gene (upstream region) where it will be negative
16
Q
What is the first base on the template transcribed/sense strand copied called?
A
transcription site (labeled +1)
17
Q
By convention do we refer to the initiation site sequence on the sense strand or the template strand?
A
on the sense strand even though the template strand is transcribed
18
Q
What are Ecoli promoter consensus sequences?
A
regions within promoters that have a similar DNA sequence
19
Q
Where are Ecoli consensus sequences found?
A
centered around -35 and -10 bases
20
Q
What is a pribnow box?
A
the -10 region
21
Q
How can promoter sequences directly demonstrate RNA polymerase binding to -35 and -10 regions?
A
by footprinting technique
22
Q
Do all of the promoters have the same efficiency?
A
no efficiency can be increased or decreased
23
Q
How does Ecoli change the efficiency of promoters?
A
using variations in -35 and -10 sequences that are used for regulation of gene expression
24
Q
How is transcription initiated?
A
RNA polymerase holoenzyme binds weakly to DNA and slides along the -35 site which binds fairly tight to form a closed complex
25
What is the next step in transcription? (elongation)
RNA polymerase moves forward to the -10 site and unwinds about 17 base pairs in front of the enzyme, then rewinds it behind the enzyme and binds tightly to form open complex (transcription bubble)
26
What is the unwinding facilitated by?
supercoiling
27
What is the first nucleotide brought into active site during transcription?
almost always a purine (pyrimidine on template)
28
What does the sigma subunit do?
only required to ensure specific efficient recognition of promoter (different sigma subunits allow different promoters to be used)
29
Is initiation regulated?
yes
30
Is initiation more simple in humans or bacteria?
more simple in bacteria
31
When is the sigma subunit lost?
once several bases are incorporated
32
How many times is elongation repeated?
about 50 bases/second
33
Where does termination occur?
some distance 3' downstream of the translation termination codon (3' untranslated region)
34
What happens to RNA polymerase during termination?
RNA polymerase core enzyme is left on the DNA without a sigma subunit, not on a promoter nd not elongating a chain (cannot bind to DNA under these conditions and it dissociates from the template)
35
What are 2 mechanisms for transcriptional termination in prokaryotes?
Rho (p) dependent
| Rho independent
36
What is rho dependent and what does it need to break hydrogen bonds?
rho proteins binds to sequences (no one really knows much about these sequences) in the 3' region of the transcript and uses ATP to break hydrogen bonds holding the transcript toth e template
37
What is Rho Independent termination?
a region of that transcript fold into hairpin loops reducing bases pairing to the template
38
What is rho independent termination driven by?
a secondary structure formation of RNA transcript and thermodynamics
39
What is a characteristic of the region of transcript following the hairpin loop?
it is very uracil rich (poor hydrogen bonding to template so DNA favors rewinding)
40
How many nuclear RNA polymerases do eukaryotes have in transcription and what is unique about them?
there are 3 nuclear RNA polymerases and they are specialized for different genes
41
Where is RNA polymerase I in eukaryotes found and what does it do?
found in the nucleolus and it makes rRNA
42
What is RNA polymerase II and III in eukaryotes found and what does each one do?
RNA polymerase II and III are found in the nucleus, RNA polymerase II transcribes mRNA and RNA polymerase III transcribes tRNAs and 5S rRNA
43
Which RNA polymerase in eukaryotes is most sensitive to the mushroom alpha amanitin toxin?
RNA polymerase II is extremely sensitive, then RNA Polymerase III, then RNA polymerase I
44
RNA polymerase II core enzyme is a multisubunit protein what does that mean?
requires numerous additional proteins for efficient initiation of transcription
45
What type of genes are highly regulated?
inflammatory genes - they are turned off unless environment factors activate it
46
What type of genes make enzymes for glycolysis?
house keeping genes or constitutive genes - means they are almost always on unlike inflammatory genes
47
What takes place in initiation in eukaryotes?
mRNA promoter has a TATA box, RNA polymerase II can bind inefficiently but initiates transcription at the correct site, then additional DNA sites and associated proteins bind to these sites
48
Where is the TATA box located?
about 25 bases upstream of +1
49
What are the 7 initiation proteins found in eukaryotic transcription initiation complex?
```
Pol II
TBP- TATA binding protein
TFIIA
TFIIB
TFIIE
TFIIF
TFIIH
(TF stands for transcription factor(
```
50
What does Pol II do?
catalyzes RNA synthesis
51
What does TBP do?
recognizes the TATA box
52
What does TFIIA do?
stabilizes TFIIB and TBP binding to promoter
53
What does TFIIB do?
binds to TBP; recruits Pol II - TFIIF complex
54
What does TFIIE do?
recruits TFIIH; has ATPase and helicase activities
55
What does TFIIF do?
binds Pol II, binds TFIIB and prevents nonspecific binding of Pol II
56
What does TFIIH do?
a kinase that unwinds DNA at the promoter; phosphorylates Pol II within CTD
57
What is a newly synthesized RNA molecule called?
a primary transcript
58
What are some modifications that most transcripts in both prokaryotes and eukaryotes undergo?
```
base modifications (tRNA)
cleavage of large primary transcript into functional fragments (rRNA)
```
59
Do prokaryotes normally have introns that need to be spliced out?
no
60
Does bacteria have a nuclear membrane?
no
61
Where is the anticodon region found on tRNA and what will it pair with?
at the bottom of the clover looking structure and will basep air with the mRNA template
62
What does RNAseP do?
removes a piece at the 5' end of the tRNA clover looking structure
63
What is the piece at the 3' end replaced with?
a CCA - this is where the amino acids are going to bind
64
Why does RNA cleavage occur?
to give each RNA intermediate
65
Does cleavage of primary transcripts occur in both prokaryotes and eukaryotes?
yes primary transcripts can be cleaved to produce smaller functional fragments in both prokaryotes and eukaryotes
66
What is found in the nucleolar region of the 5 chromosomes? (13,14,15,21,22)
a lot of gene regions (tandem arrays/multiple copies of rRNA genes)
67
How are the rRNA genes that are in the nucleolar regions processed?
post transcriptionally
68
What happens in the nucleoli?
tRNA and ribosomal proteins are arranged into large and small ribosomal subunits and shipped to the cytoplasm
69
What is thought to enhance mRNA stability in eukaryotes?
Poly A (20-250 bases)
70
What is thought to enhance translation in eukaryotes?
7 methylguanosine is added to the 5' end via 5',5'-triphosphate linkages
71
Has heteronuclear RNA been processed yet?
no
72
What does the cap binding complex do?
tethers mRNA to CTD of Pol II
73
What happens once the 5' cap is tethered to the DNA polymerase introns?
they will be spliced out which is meadiated by snRNAs (U1-U6) and they form a spliceosomem complex that cuts out this piece of mRNA that was encoded by an intron but not meant to be in the final protein
74
What is at the 3' end that helps make the lariat structure?
a branched point
75
What do OH groups serve as during mRNA splicing?
nucleophiles to first form the lariat structure then join the donor and acceptor junction
76
Where is the first cut during mRNA splicing?
at the 5' branch point
77
What is the similarity between splicing and transcription?
they are happening while it is attached to RNA polymerase as it is being transcribed (they are coordinated)
78
What is done to convert the primary transcript into mature RNA?
splicing, cleavage, and polyadenylation
| processing of the ovalbumin primary transcript slide
79
Are introns or exons larger?
introns are much larger
80
What is tissue dependent?
alternative splicing
81
What is alternative splicing?
ways that different tissues can choose which introns to splice out
82
What makes calcitonin?
the C cells of the thyroid
83
What type of mutation is most common in mitochondrial transcripts?
addition/deletion
84
What does alteration usually involve?
enzamatic deamination of A or C to form inosine (interpreted as G by translational machinery) or uridine
85
Is RNA edited before translation?
some mRNA sequences are edited before translation (doesn't always precisely match the complementary DNA sequence
86
In the intestine deamination coverts cyctidine to what?
uridine
87
Where is apoB48 protein found?
in lipoproteins in the intestive
88
Where is apoB 100 protein found?
in the liver
89
Is there a 5' cap in mRNA in eukaryotes or prokaryotes or both?
only in eukaryotes
90
Is there a 3' polyA tail in eukaryotes prokaryotes or both?
only in eukaryotes
91
Does splicing occur in eukaryotes prokaryotes or both?
mostly in eukaryotes (very very rare in prokaryotes)
92
Which RNA class is almost the same in both prokaryotes an eukaryotes?
rRNA cleavage from primary transcript in both
methylation of some ribosomes in eukaryotes
methylation of some bases in prokaryotes
93
What is similar between tRNA of eukaryotes and prokaryotes?
addition of a CCA to the 3' end and base modification are exactly the same in both eukaryotes and prokaryotes
94
What are the 2 differences in tRNA of eukaryotes vs prokaryotes?
removal of 5' leader, splicing, and removal of UU of 3' end in eukaryotes cleavage from primary transcript in prokarytoes
95
What is rifamycin (rifampicin) and what does it do?
an antibiotic that binds to beta subunit of RNA polymerase of bacteria and blocks initiation of transcription - used to treat tuberculosis
96
What is alpha amanitin?
toxin from poisonous mushroom amanita phalloides which affects RNA polymerase II and blocks mRNA synthesis
97
What is actinomycin D?
planar molecule intercalates into double helix between two GC base pairs- deforms helix blocks polymerase movement - used in cancer treatment
98
What is acridine?
research reagent that works in similar manner (also mutagen causes insertion deletion mutations
