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Biochem 3382 > Heinemann Final > Flashcards

Heinemann Final Flashcards

1
Q

What are 2 “extra” amino acids?

A

selenocysteine and pyrolysine

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2
Q

What are ribozymes?

A

segments of RNA that display enzyme activity in the absence of protein

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3
Q

What are abzymes?

A

antibodies raised to bind the transition state of a reaction of interest

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4
Q

What part of the ribosome has the catalytic activity?

A

the RNA

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5
Q

Describe the composition of the ribosome according to molecular weight

A

2/3 RNA

1/3 protein

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6
Q

Do rRNAs contain modified nucleotides?

A

yes

ie pseudouridine and ribothymidylic acid

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7
Q

What are the subunits of the prokaryotic ribosome?

A

30S and 50S

overall is 70S

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8
Q

What are the subunits of the eukaryotic ribosome?

A

40S and 60S

overall is 80S

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9
Q

What is streptomycin?

A

an aminoglycoside antibiotic
it induces mRNA misreading by binding to the small 16S rRNA of the 30S subunit of the bacterial ribosome
slows rate of bacterial growth
does NOT bind the eukaryotic ribosome

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10
Q

What is puromycin?

A

binds at the A site of BOTH prokaryotic and eukaryotic ribosomes and accepts the peptide chain from the P site
this terminates protein synthesis

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11
Q

What is found on the 3’ end of tRNA?

A

CCA

this is where the amino acid gets ligated to the tRNA

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12
Q

How long are tRNAs? Are there any modified residues?

A

73 to 94 residues each
many bases are methylated
about a 1/3 of the bases are post-transcriptionally modified

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13
Q

How is charging a tRNA with the right amino acid usually done?

A

by excluding other amino acids from the active site

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14
Q

What is one tRNA that often gets charged with the wrong amino acid?

A

proline’s tRNA sometimes gets Cys put on it

ie 1 in 100 are wrong

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15
Q

What is RNase P?

A

ribozyme
cleaves the 5’ end of tRNAs because they are longer than they need to be when they are made
(exonuclease does the 3’ end)

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16
Q

What is the hammerhead ribozyme?

A

an RNA sequence that promotes its own cleavage

we use them in the lab when we make tRNA because we don’t have RNaseP to cleave them

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17
Q

Describe Sol Spiegelman’s monster experimen

A

used RNA from a bacteriophage
its RNA replication enzyme
some free nuts and salts
the RNA started to replicate

after 74 generations the 4500 nt ended up being 218 nt (didn’t shorten again after this)

this showed that RNA could be replicated without DNA, but this still needed protein

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18
Q

Have any self replicating RNAs actually been discovered?

A

yes but only up to 14 nuts have been done and this is under lab conditions, high [ ]s etc

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19
Q

What is needed for RNA-catalyzed RNA replication?

A

ribozyme
RNA template strand
RNA primer

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20
Q

What is the RNA world hypothesis?

A

early life forms lacked protein enzymes and depended instead on enzymes composed of RNA

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21
Q

What are DNA ptotoviruses?

A

DNA genes

RNA enzymes

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22
Q

What is the origin of the nucleotides in an RNA world?

A

aminoimidazolecarbonitrile
turns into adenine

glycolaldehyde to ribose

both adenine and glycoaldehyde exist in outer space

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23
Q

What are 3 things that support the RNA world hypothesis?

A

RNA’s ability to store, transmit and duplicate genetic info
RNAs can act as enzymes
RNA genomes in viruses

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24
Q

Why does the RNA world hypothesis say that life evolved to use DNA and proteins?

A

because RNA is unstable and has poorer catalytic properties

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25
Q

What are some things against the RNA world hypothesis?

A

many of the steps needed for the nts formation do not proceed efficiently in prebiotic conditions
large RNA molecules are inherently fragile

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26
Q

What are snRNAs?

A

components of the splicesosome

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27
Q

What are snoRNAs?

A

methylation of rRNAs

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28
Q

What percentage of genes are undergoing transcription in a typical cell at and given moment?

A

3%

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29
Q

How many RNAPs do bacteria have?

A

one

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30
Q

In what direction does transcription occur?

A

polymerase moves 3’ to 5’ and synthesizes 5’ to 3’

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31
Q

What is the template strand?

A

the strand that base pairs with the new RNA i.e. the one that is being read by RNAP

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32
Q

What are the 4 stages of transcription?

A

binding of RNAP
initiation
elongation
termination

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33
Q

Explain binding of RNAP in bacterial transcription

A

subunit composition is alpha2betabeta’sigma (holoenzyme)
enzyme binds and scans along until sigma recognizes a promoter at the transcription start site which goes from -70 to +20

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34
Q

What is the closed promoter complex? Open promoter complex? What are the relative affinities of the enzyme for DNA?

A

closed = DNA is not unwound
open = DNA is unwound (12bp)
once the complex is opened the affinity is much higher

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35
Q

Describe bacterial promoters

A

~40bp on the 5’ side of the transcription start site
there are 2 consensus elements
Pribnow box near -10 with consensus TATAAT
-35 with consensus TTGACA (this is where sigma binds)

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36
Q

What determines how sigma binds to the -35 region?

A

the more the sequence corresponds to the consensus sequence the greater the efficiency of transcription

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37
Q

What is bound in the active site of all polymerases?

A

Mg2+

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38
Q

What charge does the DNA-binding cleft of RNAP have?

A

positive

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39
Q

What are sigma factors?

A

proteins that bind in the major grooves of DNA using H-bonds
there is usually one household one that does most genes and then specialized ones for different stresses i.e. heat shock
(each one recognizes a different sequence)

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40
Q

What part of the DNA does sigma interact with in bacteria?

A

-35 and -10 regions of bacterial promoters

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41
Q

Describe bacterial transcription initiation

A

ATP/GTP are preferred as the first nucleotide (this is the initiation site)
RNA synthesis starts
after 6-10 nt sigma dissociates

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42
Q

What is the elongation site?

A

the 2nd NTP

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43
Q

What is a nucleoside?

A

no phosphate

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44
Q

Describe bacterial transcription elongation

A

core polymerase (i.e. without sigma) is the elongation enzyme
it moves ~20-50 bases/sec (slower in GC-rich regions)
is very accurate
while it is occurring gyrase introduces negative supercoils infront and topoisomerase removes then behind

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45
Q

What is DNA footprinting?

A

a way to find DNA that is covered with proteins

take DNA and protein mixture and just DNA mixture
digest with DNase
run on a gel to find where the protein was binding

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46
Q

Describe chain Rho-dependent termination in bacterial transcription

A

“tethered tracking”
Rho moves along with RNAP but is slower so it trails behind unless RNAP stalls

Rho recognizes rut, closes around RNA and advances toward RNAP

when RNAP slows down in a
GC-rich region Rho catches up and causes RNAP to dissociate because it induces a change in conformation in it

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47
Q

Describe intrinsic termination of bacterial transcription

A

there are inverted repeats at termination sites that are rich in GC and forma stable stem-loop structure in the transcript
the GC-rich area slows down RNAP
then there is a run of 6-8 As, and the Us in the transcript destabilize RNAP and it dissociates

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48
Q

What is “I” in an operon?

A

the regulatory gene i.e. the one that turns the operon on and off
isn’t actually part of the operon, doesn’t need to be close to the operon but it can be

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49
Q

Where is the operator of an operon located?

A

can be upstream or downstream

usually close to the promoter sequence, but doesn’t need to be

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50
Q

What are the genes in the lac operon used for?

A

LacY is a permease, brings lactose into the cell
LacZ is a galactosidase, it isomerizes lactose to allolactose
LacA transfers an acetyl group from acetyl-CoA to beta-galactoside

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51
Q

What are the 2 major regulators of the lac operon?

A

lactose- induction

glucose- repression

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52
Q

How many promoters are there in the lac operon?

A

2

one for the structural genes and one for lacI

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53
Q

Describe the lacI promoter

A

not regulated
promoter is very weak
LacI is a tetramer, only ~10 tetramers per cell, but this is enough

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54
Q

Where does LacI bind?

A

it has 3 operator sequences
there is one strong one upstream of lacZ
and 2 weaker ones on either side of it
these operators are palindromic

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55
Q

Explain how LacI regulates the lac operon

A

when lactose isn’t present LacI binds to the operator and represses transcription
when lactose is present it gets converted to allolactose
allolactose binds to LacI and it can no longer bind to DNA
this allows transcription of the operon

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56
Q

Describe LacI and its domains

A

has a helix-turn-helix motif that binds to the major groove of the operator sequence
has allosteric site that binds the inducer
has an oligomerization domain because it functions as a tetramer
when allolactose binds there is a change in conformation and the H-T-H domains can’t interact with DNA anymore
(inducer decreases the affinity of the repressor for DNA)

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57
Q

Is the lac repressor a positive or negative regulator of the lac operon?

A

negative

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58
Q

What is negative control? What is positive control?

A 
negative = on unless turned off 
postive = off unless turned on
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59
Q

What is CAP/CRP?

A

catabolite activator protein
dimer
N-terminus binds cAMP, C-terminus binds DNA
interacts with 27 to 30 bp of DNA
increases the affinity of RNAP to the lac promoter because it is weak

60
Q

How does CAP work to increase transcription?

A

it binds upstream to the promoter to CAP sites
it interacts with RNAP through the alpha subunit and helps it bind tightly and recognize the promoter sequence
(assists in formation of the closed promoter complex)

61
Q

When is cAMP low? When is it high?

A

cAMP is low when glucose is high
and high when glucose is low
i.e. when there is no glucose want it to bind to and activate CAP
(glucose entering the cell inhibits adenylate cyclase)

62
Q

What are the binding sites for CAP?

A

TGTGA and TCANA

63
Q

Can the lac operon be transcribed when glucose and lactose are both present?

A

yes because the repressor isn’t there, but there will be very low levels of protein produced because CAP can’t bind

64
Q

How is the lac operon often used in the lab?

A

put the lac promoter in front of a gene that you want to express
add IPTG, a lactose analog when you want to induce transcription of your gene when the cells have grown enough to be ready to produce lots of protein

65
Q

What are the structural genes in the trp operon used for?

A

proteins true through trpA (five of them)

that are used in the synthesis of L-Trp

66
Q

How does the Trp repressor work?

A

under negative regulation
when trp levels are high trp binds to TrypR which allows it to bind to the operator and reduce the rate of transcription 70-fold

67
Q

What is trpL?

A

encodes the leader peptide

68
Q

What operon is TrpR from? How is it regulated?

A

TrpR comes from the torpor operon
it regulates its own operon
this is called autogenous regulation

69
Q

What is transcription attenuation? Can eukaryotes do it?

A

posttranscriptional gene repression

only bacteria can do it because transcription and translation are coupled in them

70
Q

What is the leader peptide of the trp operon?

A

139 nt segment of the operon that is transcribed under high W levels

71
Q

Which segments of the trp operon transcription can base pair?

A

1: 2
2: 3 - anti-terminator
3: 4 - terminator

72
Q

Explain attenuation in the trp operon

A

there are 2 UGG (W) codons together
when W levels are high the ribosome does not stall at them so the ribosome covers sequence 2 before 3 is transcribed
sequences 3 and 4 then base pair and form the terminator
when W levels are low the ribosome stalls at WW and RNAP keeps transcribing so the 2:3 anti-terminator sequence can form
the 2:3 structure is favourable to the 3:4 structure so if it can form it will
this can repress transcription 10-fold
(giving a total of 700-fold)

73
Q

What is a solenoid?

A

6 nucleosomes per turn

74
Q

What proteins make up nucleosomes? What influences the structure of nucleosomes?

A

4 different histones, 2 of each

modifications on histone tails, also nucleosomes can influence each other

75
Q

How do chromatin-remodelling complexes work?

A

they require ATP hydrolysis

they move nucleosomes so that DNA is accessible to RNAP

76
Q

Explain the covalent modification of histones

A

acetylated by histones acetyltransferases, this removes the positive charge from the histone so it has a lower affinity for DNA

phosphorylation of Ser, Tyr and Thr
methylation of Lys
both reduce the affinity

ubiquitination and sumolyation
reduce the affinity bc they’re in the way and bc there are residues that can repel them

there are also accessory proteins that can make the DNA bind more

77
Q

What is needed to start eukaryotic transcription?

A

transcription activators recruit chromatin-remodeling complexes and histone-modifying enzymes
then the DNA is accessible to transcription factors etc

78
Q

How many types of eukaryotic promoters are there? How are they different from prokaryotic promoters?

A

3 because there are 3 polymerases

they use transcription factors, enhancers, repressors etc which can be far away

79
Q

What do the eukaryotic RNAPs transcribe?

A 
I = pre-rRNA 
II = mRNA (most snRNA and miRNA)
III = tRNA (some rRNA and others) 
IV = siRNA in plants 
V = RNAs involved in siRNA-directed heterochromatin formation in plants
80
Q

What is amanitin?

A

poison from a mushroom that inhibits RNAP II

81
Q

Describe the general structure of polymerases

A

palm has the catalytic activity

fingers and thumb clasp the DNA

82
Q

Explain the subunit of RNAP II

A 
(all start with RPB)
1 = YSPTSPS C-terminal domain (like beta')
2 = NTP binding (like beta)
3 = core assembly (like alpha)
4 = promoter recognition (like sigma)
5-12 are all essential except for 9
83
Q

Describe the C-terminal domain of RNAP II

A

projects away, isn’t part of the core enzyme
has multiple YSPTSPS repeats, 5 of which can be phosphorylated
low phosphorylation is used for initiation
high phosphorylation is needed for elongation
this could be how it knows to start elongating

84
Q

What is the TATA box?

A

TATAAA
eukaryotic promoter that is very well conserved
it is where general transcription factors bind
TATA binding protein (TBP) binds here

85
Q

What are enhancers?

A

upstream activation sequences
they can be far away from the promoter
DNA loops and this allows multiple proteins to bind DNA sequences

86
Q

What are response elements?

A

they are promoter modules in genes that are responsive to common regulation
ie HSEs
they are found in promoter regions

87
Q

What elements are often involved in constitutive expression of eukaryotic genes?

A

TATA and GC boxes

BLEs (basal level expression)

88
Q

What is in the eukaryotic transcription initiation complex?

A

RNAP II
5 general transcription factors (GTFs)
a 20-subunit complex called Mediator (Srb/Med)

if there aren’t TFs there is no binding of RNAP (like sigma in bacteria)

89
Q

What do transcription factors do?

A

they bind to promoter elements and recruit RNAP II

90
Q

Describe formation of the pre-initiation complex in eukaryotic transcription

A

TFIID which contains TBP binds to DNA
TFIIA stabilizes it and TFIIB orients TBP
TFIIF recruits RNAP II
TFIIE recruits TFIIH (helicase)

91
Q

What is the minimal pre-initiation complex?

A

TBP, TFIIB, TFIIE, TFIIF and TFIIH

92
Q

What does TBP do?

A

other GTFs will not bind unless it does

it kinks and partially unwinds DNA

93
Q

What does Mediator do?

A

required for transcription activation
it is a bridge between gene-specific activators bound to enhancers and RNAP II/GTF machinery at the promoter

transcription co-activators bind to enhancers and recruit it, bind to its tail, it then promotes the binding of GTFs and RNAP II at its head

can also act as a repressor and cause the GTFs and RNAP II to dissociate

94
Q

Explain elongation in eukaryotic transcription

A

RNAP II pauses shortly after transcription initiation to wait for DSIF and NELF
about 25-60 nts in, basically waiting for a signal to keep going
the pause release is triggered by P-TEFb which phosphorylates CTD of RNAP II
phosphorylation causes NELF to dissociate and DSIF to turn into a positive elongation factor
then elongation can continue

95
Q

What is abortive initiation?

A

dissociation of the transcription complex energetically competes with the elongation process
can return to open promoter complex

96
Q

How is eukaryotic transcription termination thought to happen?

A

no termination sequences have been identified

there are 2 models, torpedo and allosteric

97
Q

Explain the torpedo model of eukaryotic transcription termination

A

mRNA gets cleaved after polyA signal sequence
RNase digests the remaining RNA
this disrupts the complex and RNAP dissociates

98
Q

Explain the allosteric model of eukaryotic transcription termination

A

RNAP II becomes less protective after the poly-A signal sequence

99
Q

How do activators work to activate eukaryotic gene transcription?

A

multiple activators may be required to switch a gene on
they can function synergistically
they bind to the enhancer and mediator

100
Q

What are the 3 ways activators can function synergistically?

A
  1. direct interaction i.e. they touch
  2. interaction by a bridged protein that does not contact DNA
  3. through recruitment of a chromatin modifier i.e. ones binds a modifier which modifies chromatin and allows another activator to bind
    or binding of one can disrupt the nucleosome that is blocking the binding of the another
101
Q

Why use synergy for gene expression?

A

allows for the integration of signals from more than one pathway at once i.e. different stressors at the same time

102
Q

What is combinatorial gene expression? What organisms have it?

A

refers to one gene having multiple activators

happens in both eukaryotes and bacteria, is extensive in eukaryotes

103
Q

How does transcription repression work in eukaryotes?

A

does NOT block the promoter like in bacteria
repressors can either affect the activity of an activator i.e. block it from binding or it can affect RNAP directly or indirectly through other factors
ie repressors bind to enhancer or repressor sequences but not the promoter
could cause a conformational change in RNAP II, bind to nucleosome and recruit histone deacetylase etc

104
Q

Explain how repressors can inhibit the activity of activators

A

either competitive i.e. compete for binding sites which are overlapping
or inhibition by binding to the activation domain of the activator (i.e. inactivate it instead of stopping it from binding to DNA)

105
Q

How are the GAL genes regulated in yeast?

A

the genes all contain an upstream activating sequence (UAS) that is 17bp
it is a binding site for a transactivator called Gal4p
ie UASgal is an enhancer

106
Q

What is Gal4p?

A

a transactivator
postive regulatory protein for Gal gene expression
homodimer
resemebles a Zn finger, but instead of 2 Cys and 2 His it has 6 Cys coordinating a Zn

has a DNA binding domain (N) and an activation domain (C)

107
Q

How does Gal4p regulation work?

A

when there is NO galactose:
Gal4p binds to UAS
Gal80p binds Gal4p
functions as a co-repressor and covers the activation domain of Gal4p

when there IS galactose:
Gal3p binds galactose and enters the nucleus
Gal3p binds Gal80p causing it to bind below Gal4p
Gal4- can then activate transcription because its activation domain is free

108
Q

How are bacterial inducers different from the way Gal4p activates transcription?

A

bacterial inducers have ligand binding and DNA binding on the same protein
in this case the inducer is being sensed by a different protein (Gal3p)

109
Q

How can the GAL genes be repressed?

A

when glucose is present don’t need them
Mig1 is a repressor that binds between UAS and GAL1
it recruits a repressor complex Tup1
Tup1 may recruit histone deacetylases and/or interact directly with transcriptional machinery

Note: this repression overrides induction!

110
Q

Where does gene silencing occur? What is it?

A

on CpG islands which are commonly in 5’ promoter regions
methylation of the Cs prevents transcription
methyl binding domain (MBD) proteins recognize Me-C in CpGs

111
Q

Name 4 things that DNA methylation is essential for

A

genomic imprinting
X-chromosome inactivation
suppression of repetitive elements
carcinogenesis

112
Q

What is anhydrotic dysplasia?

A

a disease that results in the absence of sweat glands

the X-linked variant can result in mosaicism in females

113
Q

How does genomic imprinting work?

A

epigenetic marks are removed in the fertilized egg

waves of de- and re-methylation occur as normal reprogramming of cells

114
Q

Explain non-specific protein-DNA interactions (in-direct readout)

A

base-independent

interact with the backbone, phosphates, aromatic base stacking

115
Q

Explain specific protein-DNA interactions (direct readout)

A

glutamine can recognize A, makes 2 H bonds with the A

arginine can recognize G and make 2 H bonds with it

116
Q

How do most proteins recognize and bind to their specific DNA sequence?

A

usually a combination of non-specific and specific interaction
ie non-specific helps make binding tighter and then specific can recognize consensus sequence

there can also be H bonding and salt bridges
proteins also have a 3D shape that is complementary to the DNA

117
Q

What is KD equal to?

A

KD= Kd/Ka

118
Q

How does EMSA work?

A

measure the strength of protein-DNA interactions
DNA is labelend with P32
DNA that is bound to protein won’t move as far on the gel
keep increasing [protein]
plot [protein] vs fraction bound
can find the KD

119
Q

What are the 3 most common DNA-binding protein motifs?

A

helix-turn-helix
Zn finger
leucine zipper basic region

120
Q

Describe the helix-turn-helix motif

A

alpha helices fit into the major groove of B-DNA
AA side chains can read the bases
alpha helix diameter is 1.2nm, major groove is 1.2nm wide and 0.6-0.8nm deep
there are 3 alpha helices in the structure but only 2 are relevant to reading the DNA
3 fits in the groove, 2 stabilizes it, 1 binds other domains etc

121
Q

What is an example of a helix-turn-helix motif?

A

homeobox domain proteins
lac and trp repressors
C-term of CAP

122
Q

Describe Zn-finger motifs

A

either C2H2 or Cx
Cys and His are coordinated to Zn
C2H2 have Cys-X2-Cys-X12-His-X3-His domains that are separated by at least 7 or 8 (up to 12) AAs
Zn finger interacts with the major groove of DNA, can read about ants
can be repeated 13 times to read many bases

123
Q

Describe bZIP

A

basic region leucine zipper
28-residue sequence with every 7th position a Leu and a basic region
every other “step” has a Leu so they stack
there are 3.6 residues per turn in an alpha-helix
leucine zippers dimerize, can be hetero or homo

Note: the leucine zipper doesn’t recognize the DNA sequence, they are the dimerizaion domain
the basic region it what contacts the DNA
Myc, Fos and Jun are examples

124
Q

How is 5’ capping of eukaryotic mRNA done?

A

capping enzyme is bound to RNAP II
reaction is catalyzed by a guanylyl transferase
cap is added when the RNA is about 25-30nt long i.e. during transcription
5’-5’ triester linkage
gets methylated at position 7 after it is added
additional methylations occur at 2’OH of the next 2 residues and 6-amino of the first A

125
Q

What are the functions of the 5’ cap on mRNA?

A

protection from degradation
enhancement of translatability
transport out of the nucleus
proper splicing

126
Q

Where do the phosphates in the 5’-5’ linkage of the cap in eukaryotic mRNA come from?

A

2 are from the transcript, 1 is from the GTP

127
Q

What is Cap O? Cap 1? Cap 2? Which is most common

A

Cap O = only guanyl methylated
Cap 1 = methyl also on 2’OH of first nt
Cap 2 = methyl on 2nd nt

Cap O is always present, Cap 1 usually is
(Cap 1 is the most common)

128
Q

What molecule methylates 5’ caps?

A

S-adenosyl-L-methionine:mRNA (guanine-N7-)-methyltransferase
methyl group comes from SAM

129
Q

What are the functions of 3’ adenylation?

A

aids in transcription termination directs export of the mRNA and translation
protects 3’ end from degradation
helps determine half life (when tail gets short enough the mRNA is degraded)

130
Q

What is the polyA addition site? What is the other signal?

A

AAUAAA

GU-rich region downstream of the polyA signal

131
Q

Explain how polyadenylation occurs

A

CPSF binds to AAUAAA
CStF binds to GU-rich region and CPSF, forming a loop in the pre-mRNA
CFI and CFII stabilize it
PolyA polymerase (PAP) cleaves the strand 10-30 nts downstream of the AAUAAA
cleavage factors are released
PAP slowly begins to create a polyA tail (does not need a template)
PABII and other proteins bind to the nascent tail and this increases the speed of polyadenylation
PABII tells PAP to stop and it dissociates
Note: PABPs usually stay on the mRNA while it is exported to the cytosol and help protect it there

132
Q

What is alternative polyadenylation?

A

one gene can have more than one polyadenylation site
can change the resulting protein
which signal is used depends on whether the factors are present and their affinity for the RNA

133
Q

How are mRNAs often purified?

A

use polled primers attached to magnetic beads

hold a magnet and wash out everything else

134
Q

Explain RNA polyadenylartion in bacteria

A

PROMOTES mRNA degradation by the degradosome (polynucleotide phosphorylase and RNase E)
polyA tail changes the secondary structure and allows polynucleotide phosphorylase to bind to the 3’ end
also recruits RNases
polyA tail is only ~30nt long

135
Q

Describe RNA export to the cytoplasm

A

PABP promotes export from the nucleus and translation and inhibits degradation

mRNAs that aren’t exported are degraded by the exosome

136
Q

Which bases in RNA get deaminated? What do they pair with?

A

A to I, will pair with G

C to U, will pair with A

137
Q

How is ApoB RNA edited?

A

C to U by ApoBEC-1 in the small intestine at residue 2153 generates a stop codon, UAA

138
Q

Where does splicing occur?

A

in the nucleus

after it has been capped and polyadenylated and proteins that keep the pre-mRNA untangled have bound

139
Q

What are longer, exons or introns?

A

introns

140
Q

What is the 5’ splice site consensus sequence?

A

Exon: AG
Intron: GUAAGU

141
Q

What is the 3’ splice site consensus sequence?

A

Intron: Py (8) -CAG
Exon: G –
there is also an A branch site near the pyrimidine-rich region, 10-50 bases from the splice site

142
Q

Explain how the spliceosome works

A

U1 subunit binds to the GU at the 5’ end of intron (contains nRNA base pairing with GU site)
U2 binds to A-branch site (through nRNA but not paired with the A)
complex brings the 5’GU to the pyrimidine-rich region
U4 and U6 snRNPs lock U1 and U2 in, are independent of the pre-mRNA sequence
U5 joins, the spliceosome is fully assembled which induces a conformational change and starts splicing
5’ end is cleaved using ATP hydrolysis for energy
intron is looped back and 5’ end and A-site form a new ester bond making the branch lariat structure
consensus sequence for the branch site is YNYRAY (y =pyr, r= pur)
3’ end is then cleaved
ester bond between 3’ end of intron and 5’ end of exon exchanges for bond between the 2 exons
ligated exons are released, intron is still bound to spliceosome
lariat is released and degraded by a deb ranching enzyme

143
Q

What is part of the spliceosome?

A

snRNPs and pre-mRNA

144
Q

What is constitutive splicing?

A

every intron is removed and every exon is incorporated into the mature RNA

145
Q

Where are self-splicing introns found?

A

note: they are ribozymes!

in some bacteria and organelles

146
Q

How do group 1 self splicing introns work? Where are they found?

A

protists, bacteria, bacteriophages

exogenous G is recruited
it does a nucleophilic attack on 5’ exon/intron boundary
forms phosphodiester linkage with 5’ end of intron
5’ exon OH attacks 3’ boundary linking the 2 exon
linear intron is released

147
Q

How do group w self splicing introns work? Where are they found?

A

fungal and land plant mitochondria, algal plastids, bacteria, archaea

2’OH group of an intramolecular adenosine attacks 5’ splice site and forms a lariat structure
freed 5’ exon OH attacks 3’ splice site
lariat intron is released

Biochem 3382 (4 decks)

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