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Chapter 31
Transcription
1.
2.
3.
4.
The role of RNA in protein synthesis
RNA polymerase
Control of transcription in eukaryotes
Posttranscriptional processing
Three major classes of RNA
All participate in protein synthesis:
• Ribosomal RNA, rRNA
1. Transfer RNA, tRNA
2. Messenger RNA, mRNA
All are synthesized from DNA by
transcription
Historically
DNA found in cell nucleus, but RNA found in
cytosol (1930), microscopy and cell fractionation
Concentration of cytosolic RNA-Protein particles
correlate with protein synthesis - is site of
protein synthesis - was later identified as
Ribosome
In eukaryotes, DNA is never in association with
protein synthesis (Ribosome)
Incorporation of radiolabelled amino acids occurs in
association with RNA-Protein particles
Structure of DNA revealed possible copy
mechanism
Central Dogma
DNA
RNA
Protein
1. The role of RNA in protein
synthesis
Studied by following enzyme induction
Bacteria vary the synthesis of certain
enzymes depending on environmental
Conditions
Enzyme induction occurs as consequence
of mRNA synthesis
Enzyme induction
E. coli can synthesize ˜4300 polypeptides
But enormous variation in abundance of
specific polypeptides:
Ribosomal protein: 10’000 copies/cell
Regulatory protein: <10 copies/cell
Housekeeping enzymes, constitutive
Adaptive, inducible enzymes
Lactose-metabolizing enzymes are
inducible
E. coli is initially unable to metabolize
lactose, but starts to induce the
corresponding enzymes:
lactose permease, for uptake
β-galactosidase, for splitting lactose
Few copies -> >10% or proteins, 1000-fold
Triggering substance: inducer, allolactose
or IPTG
The induction kinetics of βgalactosidase in E. coli
The E. coli lac operon
Structural genes
Z, β-galactosidase
Y, lactose permease
A, thiogalactisodase transacetylase
Control site
P, promoter
O, operator
Regulatory gene
I, inducer
Bacteria can transmit
genes via conjugation
Conjugation allows for bacterial genetics
Constitutive mutation: lac operon induced
even without inducer -> mutation in I,
distinct but closely linked to structural
genes
PaJaMo experiment, 1956, Arthur Pardee
Francois Jacob, Jacques Monod
Bacterial conjugation
F– cell acquires an F
factor from an F+ cell
Bacterial conjugation:
Transfer of genetic material
Between a donor cell, F+, and
recipient, FAbility to conjugate/mate is encoded
on a plasmid, F factor (fertility)
F+ cell are covered with F pili
Allows binding to F- cell surface
Formation of cytoplasmic bridge and
transfer of genetic information
Converts F- to F+
Transfer of the bacterial
chromosome from an Hfr cell
to an F– cell and its
subsequent recombination
with the F– chromosome
F factor can spontaneously integrate
into the genome -> Hfr strain
(high frequency of recombination)
-> transmission of genomic information
upon mating:
in fixed order
time dependent (90min)
Merozygote, partially diploid
Recombination and integration
The PaJaMo experiment
Mate Hfr I+Z+ to F- I-Z- in absence of inducer
Monitor β-gal activity over time
Induction after 1h, cessation upon 2h -> Z+ in I- cells leads to
constitutive induction, cessation upon transfer of I gene -> I
gene codes for diffusible repressor of Z, lac repressor
F- is resistant to T6 and streptomycin
Messenger RNA
Second type of constitutive mutation Oc, operator
constitutive, maps between I and Z genes
In merozygote F’ Oc Z- / F O+ Z+, β-gal inducible
But in Oc Z+ / F O+ Z- constitutive synth of β-gal
O Can control Z only when on same chromosome !!!
-> cis acting control
I is trans acting factor
Proteins are synthesized in two stages:
1. DNA is transcribed in mRNA
2. mRNA is translated into protein
This model explains behavior of lac system
Messenger RNA
In the absence of inducer, I binds to O and
represses synthesis of structural genes Z, Y, A
On binding inducer, repressor dissociates from O,
permitting transcription and subsequent translation
Operator-Repressor-Inducer system represents a
molecular switch
Oc is constitutive because repressor cannot bind,
Cis-acting element
Coordination of all 3 protein by single polycistronic
mRNA transcript, cistron
mRNAs have their predicted
properties
Kinetic of enzyme induction -> mRNA has to be
rapidly synthesized and degraded, short half-life
rRNA turnover is slower, comprises 90% of cellular
RNA
The distribution, in a CsCl density gradient,
of 32P-labeled RNA that had been synthesized
by E. coli after T4 phage infection
The hybridization of 32P-labeled
RNA produced by T2-infected E.
coli with 3H-labeled T2 DNA
2. RNA Polymerase
RNA polymerase is responsible for the DNA-directed
synthesis of RNA (1960), dNTP and DNA-dep.
E. coli RNAP hplpenzyme, 459kD, αββ'ωσ subunit
composition, sigma σ70 unit dissociates from core
once RNA synthesis has been initiated
RNAP functions:
1. Template binding
2. RNA chain initiation
3. Chain elongation
4. Chain termination
Components of E. coli RNA
Polymerase Holoenzyme
Electron micrograph of E. coli RNA polymerase
(RNAP) holoenzyme attached to various promoter
sites on bacteriophage T7 DNA
Template binding
o RNA synthesis is initiated only at specific sites on the
DNA template
o RNAP binds to its initiation sites at sequence elements
called promoter, these are recognized by sigma factor (K
≈10-14M)
o Promoter, ca. 40bp element, located 5’ of structural
gene, first base in RNA is +1, initiation site
o If RNAP bound to promoter -20 to +20 are DNaseI
protected
o Consensus promoter sequence, hexamer centered at 10 = Pribnow Box, TATAAT, plus additional element at 35 sequence in between not important, but distance
o +1 is either A or G
The sense (nontemplate) strand
sequences of selected E. coli promoters
Rate of transcription varies 1000-fold,
correlates with strength of promoter to bind RNAP
Initiation requires formation of
an open complex
RNAP binding alters accessibility of bases towards
methylating agents (dimethyl sulfate, DMS), DMSfootprinting -> holoenzyme binds to only one side/face of
DNA and melts DNA
Chain initiation
+1 is purine, A > G
Initiation reaction: pppA + pppN -> pppApN + Ppi
Unlike DNA replication, RNA initiation does not need a
primer
Crab claw shape of Taq RNAP, pincers formed by β and
β ’ with a cavern between the two pincers
Prokaryotic initiation is inhibited by rifamycin B,
Steroptomyces mediterranei, rifampicin commercial grampositive antibiotic (also tubeculosis), does not block
promoter bdg or elongation, but only initiation
Structure of Taq RNAP core
enzyme
Chain elongation
5’ -> 3’ or 3’ -> 5’ growth ?
Label with γ[32P]GTP, chase with cold GTP
If 5’->3’, RNA is permanently labelled
If 3’->5’, RNA is unlabelled
Transcription supercoils DNA
Elongation requires opening of dsDNA, bubble
2 models:
RNAP swirls around DANN -> transcript would wrap
DNA rotates -> DNA must be tethered
Transcription occurs rapidly and
accurately
In vivo rate is 20-50nt/sec, entirely processive, no exonucleolytic correction like in DNA pol.
One mistake per 10’000nt, tolerable because:
1. Genes are repeatedly transcribed
2. Genetic code is redundant -> high prob. of silent mut.
3. Aa substitutions in proteins are often tolerated
4. Large portion of transcript is non-coding (intron)
Intercalating agents inhibit
both RNA and DNA polymerase
Actinomycin D intercalates into DNA and
RNA, inhibits transcription and replication
Chain Termination
EM specific sites of termination, two common features in E.
coli: 1. Series of 4-10 consecutive A-Ts, As on template
2. G + C-rich palindromic region upstream of A-Ts
A hypothetical strong
(efficient) E. coli terminator
The stability of terminator G+C hairpin + weak base pairing
Between RNA polyU and DNA template ensure termination
Termination often requires the
assistance of Rho factor
50% of termination sites lack cis-acting terminator
sequences, but require protein factor, Rho
in vivo transcripts often shorter than in vitro
Rho is hexameric, 419Aa
Helicase activity
Recognition sequence on RNA transcript
Eukaryotic RNA polymerase
3 distinct types of RNAP
1. RNAP I, in nucleoli, makes rRNA
2. RNAP II, in nucleoplasm, makes mRNA precursors
3. RNAP III, nucleoplasm, 5S rRNA, tRNA, small RNAs
Up to 600kD, up to 12 subunits, 5 of these present in all 3
RNAP types
RNAP II has extraordinary C-terminal domain, CTD
52 repeats of PTSPSYS, 50 Ser are phosphorylated
Transcription is only initiated if CTD is unphosphorylated
Elongation occurs only if CTD is phosphorylated
Phosph. Converts initiation complex to elong. compl.
RNA Polymerase Subunitsa
X-Ray structure of yeast RNAP
II that lacks its Rpb4 and
Rpb7 subunits
Resembles Taq RNAP
crab claw like shape
Cutaway schematic diagram of the
transcribing RNAP II elongation
complex
On DNA binding, 50kD clamp
swings out -> processivity
Amatoxins specifically inhibit RNA
polymerase II and III
Poisonous mushroom, Amanitia phalloides
Responsible for majority of fatal mushroom poisonings
Toxin, bicyclic octapeptide, amatoxins, α-amanitin
Tight 1:1 complex with RNAP, K 10-8M
Act slowly, death after a few days -> turnover of RNA
Mammalian RNAP I has a bipartite
promoter
Numerous rRNA genes have essentially identical sequence
and promoter
But unlike RNAP II and RNAP III, RNAP I promoters are
Species specific !!
Core promoter -31 to +6
and upstream element (-187 to -107)
RNAP II promoters are complex
and divers
Euk RNAP II promoters are more complex than their
prokaryotic homologues
GC-box upstream of constitutive genes
Selectiveley expressed genes often contain TATA box
(-27 to -10), resembles -10 of prok. Genes
Mutation sin TATA box cause heterogeneity in initiation
CCAAT box, -70 tp -90, i.e. in globin genes
The promoter sequences of selected
eukaryotic structural genes
Enhancers are transcriptional activators
that can have variable positions and
orientations
Promoter element that act in both orientation and distance
independent = enhancers
Can act from several kb, in euk. Viruses or structural genes
Required for full activity of promoter
Recognized by specific transcription factors -> DNA loop
Stimulate entry of RNAP II on promoter
Mediate much of selective gene expression
RNAP III promoters canbe located
downstream from their transcription
start site
RNAP III promoters can be within the gene’s transcribed
region !! 5S rRNA
3. Control of Transcription in
Prokaryotes
Adaptation to environmental
changes takes only minutes
because transcription and
translation in prokaryotes are
coupled (euk. takes hours)
mRNAs are degraded in 1-3min
Promoters
Genes that are transcribed at high rates have efficient
promoters
Lac I is transcribed at 10 copies/cell
Gene expression can be controlled
by a succession of sigma factors
Cell development and differentiation involves the
temporally ordered expression of specific sets of genes
according to a genetically specified program
For example phage infection in prokaryontes
1. Expression of early genes
2. Expression of middle / late genes
One way of regulation: cascade of sigma factors that
recognize the respective promoters
lac Repressor I: Binding
1966 isolation of repressor based on binding to radioLabelled IPTG, protein low abundance (0.002%)
Tetramer, 360 Aa, binds DNA K = 10-6M, promoter 10-13M
Trypsin cleavage releases two domains. N-term. binds DNA
rest binds IPTG
Protein scans DNA to bind to promoter (on rate is greater
than diffusion limited process).
lac operator has a nearly
palindromic sequence
Repressor protein used to “fish” binding DNA sequence
Lac I binds to 26bp element with nearly 2-fold symmetry
lac repressor prevents RNA polymerase from
forming a productive initiation complex
RNA polymerase binds +20 to -20
Operator occupies +28 to -7
-> lac operator and promoter overlap
Binding of repressor obstructs RNAP binding
Catabolite Repression: An example
of gene activation
Glucose is the metabolite of choice !!!
In its presence, no other C-source is being metabolized,
>100 enzymes are repressed (arabinose, galactose, lactose)
= catabolite repression
Prevents wasteful duplication of energy producing
enzymes
The kinetics of lac operon mRNA synthesis
following its induction with IPTG, and of its
degradation after glucose addition
cAMP signals the lack of glucose
cAMP is second messenger in animal cells
In E. coli, cAMP greatly diminished in presence of glucose
Addition of cAMP to culture overcomes catabolite
repression
CAP-cAMP complex stimulates the
transcription of catabolite repressed operons
cAMP binding protein = catabolite gene activator protein,
CAP = cAMP receptor protein, CRP
Homodimer of 210 Aa, undergoes large conformational
change upon cAMP bdg.
CAP-cAMP binds lac operon and stimulates transcription ->
positive regulator (unlike lac I, negative regulator)
Binds DNA and bends it 90°, contacts CTD of RNAP
X-Ray structures of CAP–cAMP
complexes
Sequence-specific protein-DNA
interactions
Genetic expression is controlled by proteins such as CAP,
lac repressor
How do proteins bind to specific DNA sequences, how do
they recognize base (pairing) ?
Base position in minor (5Å wide, 8Å deep groove is sequence
independent, but varies in major groove !
-> protein / base recognition via major groove, 12Å wide,
8Å deep
The helix-turn-helix is a common DNA
recognition element in prokaryotes
Cap dimer’s two symmetrical F helices fit into two successive
major grooves of B-DNA
CAP’s E and F helix form a helix-turn-helix (HTH) motif
(supersecondary structure), similar to lac repressor, trp
repressor
HTH is 20 Aa motif, helices cross at 120°
F helix in CAP is recognition helix, complex structural
interactions (hydrogen bonds, salt bridges and van der Waals
interactions), i.e. no simple Aa - Base code
HTH-DNA interaction
araBAD operon:positive and negative
control by the same protein
Arabinose is not metabolized by human, but E. coli in our gut
will metabolize this pentose
5 enzymes form an catabolite repressible araBAD operon
Control sites araI, araO1, araO2
Regulator: araC, homodimer 292Aa, N-term arabinose binding
and dimrization domain, linker + C-term DNA bdg. domain
Genetic map of the E. coli araC
and araBAD operons
Mechanism of araBAD regulation
lac repressor II: structure
DNA loop formation is an important mechanism for
transcriptional regulation
The lac repressor is a dimer of dimers, V-shaped
Model of the 93-bp DNA loop formed
when lac repressor binds to O1 and O3
DNA loop is further
stabilized by cAMP-CAP
binding
Principal:
Modular build up and
break down of very
high affinity complexes
trp operon: Attenuation
Attenuation: Control mechanism to regulate amino acid
biosynthetic operons
E. coli trp operon: five polypeptides, 3 enzymes,
mediate the synthesis of trp from chorismate
Regulated by trp repressor, homodimer, 107Aa,
bind L-trp -> binds trpO -> repression
Trp acts as a corepressor
Genetic map of the trp operon
Tryptophan biosynthesis is
regulated by attenuation
trpE, first structural gene in trp operon is preceded by
trpL, 162nt leader sequence
Availability if trp results in premature transcription
Termination within trpL
Control element for this transcription termination =
attenuator
trpL contains 2 consecutive trp codons thereby couples
Translation rate to formation of RNA secondary structure
and transcription termination
Similar in his operon, ilv operon
The alternative secondary
structures of trpL mRNA
The trp attenuator’s transcription
termination is masked when trp is scarce
Amino Acid Sequences of Some
Leader Peptides in Operons Subject
to Attentuation
Regulation of rRNA synthesis:
The stringent response
E. coli division 20 min, contains 70’000 ribosomes
-> must synthesize 35’000 ribosomes/20 min
Initiation of rRNA transcription : 1 /sec -> 1200
ribosomes/20min
⇒Seven distinct rRNA operons per chromosome
+ multiple replicating chromosomes
Coordination: rate of rRNA synthesis proportional to rate
of protein synthesis
Molecular control of this coordination: Stringent Response
(p)ppGpp mediates the stringent
response
o The stringent response is correlated with a rapid intracellular
accumulation of two unusual nucleotides:
ppGpp and pppGpp = (p)ppGpp
o Rapid decay when amino acids become available
o relA- mutants exhibit no stringent response = relaxed control lack
(p)ppGpp
o (p)ppGpp inhibits the transcription of rRNA genes, but
stimulates transcription of trp and lac operons
o (p)ppGpp alters RNAP promoter specificity
o Rel A, stringent factor: ATP + GTP <-> AMP + pppGpp
o Active in association with ribosome engaged in translation but lack
charged tRNAs
o (p)ppGpp degradation by SpoT
4. Posttranscriptional Processing
Primary transcripts - of eukaryotes - are not yet
functional but undergo post-transcriptional
modifications:
1. Exo- and endonucleolytic removal of nt
2. Appending nt at 3’ and 5’ ends
3. Modification of specific nucleosides
Messenger RNA processing:
caps, tails, and splicing
In eukaryotes: primary transcripts are made in the
cell nucleus, but translation takes place in the cytosol
Primary transcripts are processes on their transport way
to the cytosol
Eukaryotic mRNAs are capped
Cap: 7-methylguoanosine is joined
at 5’ nucleoside via
5’-5’ triphosphate bridge
Cap defines eukaryotic translation
start site
Addition requires:
1. RNA triphosphatase
2. Capping enzyme
3. Guanine-7-methyltransf.
4. 2’-O-methyltransf.
Eukaryotic mRNAs have poly(A) tails
Unlike prokaryotic mRNAs, eukaryotic mRNAs are
always monocistronic.
Termination process in imprecise -> heterologous 3’
ends, but mature mRNAs have well defined 3’ends
with tails of ~250 polyAdenosine nucleotides
Appended by two reactions:
1. Cleavage of (heterologous) 3’ end, ~20nt
past AAUAAA sequence; CFI, CFII
2. Poly(A) polymerase, PAP
Poly(A) tail gets shorter as mRNA ages
Mature histone mRNAs lack poly(A) tail
Eukaryotic genes consist of alternating
expressed and unexpressed sequences
Primary transcripts are heterogenous in size and much
larger than mature mRNAs !! ->heterogenous nuclear
RNA (hnRNA) mature to mRNAs by excision of internal
sequences (pre-mRNA)
Intervening sequence = intron (~1500nt, average 8/gene)
Expressed sequence = exon (~300nt)
Largest gene, titin 29’926 Aa, 234 introns, 17kb exon
Exons are spliced in a two-stage
reaction
Gene splicing must be precise to maintain the reading
frame !!
The consensus sequence at the
exon–intron junctions of vertebrate premRNAs
Invariant GU at intron 5’ boundary, AG at 3’ boundary
The sequence of transesterification reactions
that splice together the exons of eukaryotic
pre-mRNAs
Types of Introns
Exons are spliced in a two-stage
reaction (2)
1. Formation of 2’,5’-phosphodiester bond between
adenosine in intron and 5’ phosphate
-> intron assumes lariat structure
2. Free 3’-OH of 5’ exon generates phosphodiester
with 3’ exon, -> releasing the intron lariat
Intron lariat is then debranched, and degraded
Note. No free energy input !
Some eukaryotic genes
are self-spliced
Today we know 8 distinct types of
introns:
Group I introns, nuclei,
mitochondria, chloroplasts
Tetrahymena, ciliate, no protein
required for splicing, RNA only
+ guanosine
Self-splicing RNA = ribozyme
Group II introns
Mitochondria of fungi and plants
Self splicing, lariat intermediate but no external
nucleotide
Spliceosome is an RNA-protein complex that mediates
splicing of normal pre-mRNA,
evolved from group primordial self-splicing RNA,
Protein thought to be important for fine-tuning of
ribozyme structure,
Similar, RNA of ribosome has catalytic activity
=> RNA world hypothesis
The self-splicing group I intron
from Tetrahymena thermophila
Hammerhead ribozymes catalyze an
in-line nucleophilic attack
Simplest and best characterized ribozymes
Embedded in the RNA of certain plant viruses
Termed hammerhead enzyme due to structural resemblance
Enzyme green
Substrate blue
Cleavage site red
Splicing of pre-mRNAs is mediated
by snRNPs in the spliceosome
o How are splicing junctions recognized and how are the
two exons joined ?
o Eukaryotes contain many 60-300nt nuclear RNAs,
termed small nuclear RNAs, snRNA
o Form RNA-protein complexes termeds small nuclear
ribonucleoproteins, snRNPs
o U1-snRNA (U-rich) is complementary to 5’ splice site,
recognizes this splice site
o Splicing takes place in 45S particle, spliceosome, which
brings together pre-mRNA and snRNPs, 5 RNAs, ~65
proteins, ATP-dep., U2-,U4-, U5-, U6-snRNPs
Page 1264
Figure 31-55
The X-ray
structure of the catalytic
pocket in the hammerhead
ribozyme’s kinetically trapped
intermediate.
An electron
micrograph of
spliceosomes in
action
A schematic diagram of six rearrangements that the
spliceosome undergoes in mediating the first
transesterification reaction in pre-mRNA splicing
1.
2.
3.
4.
5.
6.
Exchange of U1 for U6 in base pairing to 5’ splice site
Exchange of BBP for U2 in binding to branche site
Intramolecular rearrangement in U2
Disruption of pairing between U4 and U6
Disruption of a second stem between U4 and U6
Disruption of a stem-loop in U2
Splicing also requires the
participation of splicing factors
o Variety of proteins known as splicing factors that are
not part of the spliceosome also participate in the splicing
reaction
o Branche point binding protein, BBP (=SF1, U2AF)
o SR proteins (Ser, Arg-rich) and members of the
heterogenous nuclear ribonucleoprotein family (hnRNP),
contain RRM domain (RNA Recognition Motif), hnRNP are
highly abundant
o Exon skipping does not normally occur, splicing occurs
orderd in 5’->3’ direction, cotranscriptional
Structure of the RNA binding portion of
human branch point-binding protein (BBP) in
complex with its target RNA
Spliceosomal structures
o All 4 snRNPs involved in pre-mRNA splicing contain the same
snRNP core protein, which consist of 7 Sm proteins (react
with autoantibodies from patients with systemic
erythematosis), named B/B’, D1, D2, D3, E. F, and G protein
o Each of these Sm proteins contain two conserved segments,
Sm1 and Sm2 separated by a variable linker
o The seven Sm proteins bind to conserved RNA sequence,
the SM RNA motif, occurs in U1-, U2-, U4, and U5-snRNA
o Form heptameric ring, central hole positive charged allows
passage of ss RNA but not ds RNA
o U1-snRNP consist of 10 proteins, 7 Sm proteins and 3 U1
specific factors
A model of the snRNP core
protein
The electron microscopy-based structure of
U1-snRNP at 10 Å resolution
The predicted secondary structure of U1-snRNA
The molecular outline of U1-snRNP
Significance of gene splicing
o
o
o
o
o
o
Why are there introns ?
Introns are rare in prokaryotic structural genes
Uncommon in yeast, 239 introns in 6000 genes
Abundant in higher eukaryotes
Histones lack introns
Unexpressed sequences constitute 80% for a typical
vertebrate structural gene
o Molecular parasites (junk DNA) ?
o Evolution of complex spliceosome must have been
advantageous over elimination of split genes
o intron/exon organization is:
- An advantage for rapid evolution of new proteins
- Allows gene function tuning through alternative splicing
Many eukaryotic proteins consist of
modules that also occur in other proteins
o Example, LDL-receptor
839 Aa, 45kb gene, 18 exons, most encode specific
functional domains, 13 of these segments/domains have
homology with domains found in other proteins
=> modular construction of the LDL receptor
=> modular construction is found in many other proteins
that are composed of re-utilized domains (i.e. signal
transduction, SH2, SH3 domains etc.)
Alternative splicing greatly increases the number
of proteins encoded by eukaryotic genome
o The expression of numerous cellular genes is modulated by
the selection of alternative splice sites
o Example rat α-tropomyosin gene encodes 7 tissue specific
variants of the muscle protein
Alternative splicing
o Occurs in all metazoans
o Human genome only 30’000 genes but estimated 50’000 140’000 structural genes
o Entire functional domains or single amino acids can be
altered in proteins through alternative splicing
- soluble or membrane bound
- can be phosphorylated by a specific kinase or not
- subcellular localization
- whether enzyme binds a specific allosteric effector
- affinity of receptor - ligand interaction
o Selection of alternative splice sites is developmental and
tissue specific (regulation in space and time)
Selection of alternative splice sites
Best understood for Drosophila sex-determination genes
no TRA protein
functional TRA protein
(repression of splice site
by Sxl)
Male-specific DSX protein -> represses female-specific genes
Female-specific DSX protein -> represses male-specific genes
(activation fo splice site)
AU-AC introns are excised by a novel
spliceosome
o Small fraction of introns (~0.3%) have AU rather than GU
at their 5’ends and AC rather than AG at 3’
But are excised via lariat intermediate
Are splice by a AU-AC spliceosome with U5 sn RNP in
common but specific U11, U12 and U4atac-U6atac
Trans-splicing
o Trans-splicing, joining of two separate RNA molecules
- Observed in Trypanosomas, all mRNAs have same 35nt
leader, but this leader is not present in the
corresponding genes
- Splice leader (SL) RNA, transcribed from an
independent gene
- Trans-splicing reaction resembles spliceosome-mediated
cis-splicing
- But Y-shaped rather than lariat intermediate
The sequence of transesterification
reactions that occurs in trans-splicing
RNA can be edited by the insertion or
deletion of specific nucleotides
o Certain RNA differ in sequence from their corresponding
genes
Examples: C->U and U->C changes
Insertion or deletions of U
Insertion of multiple G or C residues
o Most extreme case in mitochondria of Trypanosomes
involves addition and removal of hundreds of U’s to and
from 12 otherwise untranslatable mRNAs
o RNA editing, violates central dogma ? Because not
template-based
o Discovery of guide RNAs (gRNAs), 50-70nt, 3’ oligo(U) tail
A schematic diagram indicating how gRNAs
direct the editing of trypanosomal pre-edited
mRNAs
RNA editing occurs on ~20S RNP, editosome
gRNA is used as template to “correct” the mRNA
Requires: 1. Endonuclease
2. Terminal uridylyltransferase
3. RNA ligase
Trypanosomal RNA editing
pathways
RNA can be edited by base deamination
o Humans express two forms of apolipoprotein B (apoB):
- apoB-48, only made in intestinal tissue, functions in
chylomicron transport, triacylglycerol to liver and periphery
- apoB-100, made in liver, functions in VLDL, IDL, and LDL
to transport cholesterol from liver to periphery
o apoB-100 is 4536Aa large protein, apoB-48 consists of
apoB-100 N-terminal 2152 residues but lacks C-term domain
of apoB-100 that mediate LDL receptor binding
o Both are expressed from the same gene, mRNAs differ in a
single C->U change, resulting in stop codon (UAA)
o Base change mediated by a protein, cytidine deaminase
substitutional editing, also in glutamate receptor
RNA interference
o Noncoding RNAs can have important roles in controlling
gene expression
o Anti-sense RNA can block translation of a specific message
Yet injection of sense RNA into C. elegans also blocks
protein production
o Added RNA interferes with gene expression = RNA
interference, RNAi
o 1998 Andrew Fire and Craig Mello, Nobelprice 2006
ds RNA is substantially more efficient in causing RNAi
induced by only a few molecules -> catalytic rather than
stoichometric
A model for RNA interference (RNAi)
1.
Trigger RNA is cut to 21-23nt
oligos =small interf. RNA
siRNA, with 2nt overhang at 3’ and
5’ phosphatemediated by RNase,
Dicer
2. siRNA is transfered to
multisubunit RISC complex, RNAinduced silencing, siRNA guides
substrate specificity
3. RISC cleaves mRNA, which is then
further degraded
RNAi requires that trigger dsRNA
is copied, mediated by RNAdependent RNA polymerase (RdRP)
Method of choice for knockout
study
A model for transitive
RNAi
siRNA can prime for RdRPcatalyzed synthesis of secondary
trigger dsRNAs which are diced
to yield secondary siRNAs
May yield non-specific silencing,
Transitive RNAi
RNAi may arose as defense
against RNA viruses, inhibit
movement of retrotransposons
Ribosomal RNA Processing
o Seven rRNA copies in E. coli genome,
o polycistronic >5500nt transcript, containing 16S rRNA, 1-2
tRNAs, 23S rRNA, 5S rRNA, plus 1-2 more tRNAs at 3’
o Processing into mature rRNAs, cotranscriptional
o Specific endonucleolytic cleavage by RNase III, RNase P,
RNase E, RNase F
o Secondary processing, trimming of 5’ and 3’ ends occurs
while rRNA is already associated with ribosomal proteins
The posttranscriptional
processing of E. coli rRNA
Ribosomal RNAs are methylated
o During ribosome assembly, 16S and 23S rRNA is methylated
at 24 specific nucleosides, SAM-dep.
o N6,N6-dimethyladenine and O2-methylribose (protect from
RNase degradation)
Eukaryotic rRNA processing is guided
by snoRNAs
o rRNA transcription and processing takes place in nucleoli
o Primary transcript is ~7500nt 45S RNA that contains 18S,
5.8S, 28S rRNAs separated by spacer sequences
o Specific methylation (106 sites in humans)
o Conversion of U to pseudouridine (95 in humans)
o Subsequent cleavage and trimming analogous to prok.
o How are methylation sites recognized/targeted ?
o Pre-rRNA interact with small nucleolar RNAs, snoRNAs
(~200 in mammals), intron-encoded
The organization of the 45S primary
transcript of eukaryotic rRNA
Transfer RNA processing
o tRNA, ~80nt, cloverleaf secondary structure, large fraction
of modified bases
o E. coli chromosome contains 60 tRNA genes, some are
components of rRNA operons
o Primary transcript contains 1-5 tRNA copies, excision and
trimming similar to rRNA processing
o Many tRNAs contain introns
o CCA trinucleotide to which the amino acid is appended is
postracriptionally added, tRNA nucleotidyltransferase
o RNase P generates 5’ ends of tRNAs, contains 377nt RNA
component, is catalytic subunit
A schematic diagram of the tRNA
cloverleaf secondary structure
Page 1278
Figure 31-74a The structure
of the RNA of B. subtilis
RNase P. (a) Predicted
secondary structure with
specificity domain drawn in
various colors and catalytic
domain is black.
Page 1278
Figure 31-74b The structure
of the RNA of B. subtilis
RNase P. (b) The X-ray
structure of the specificity
domain in which its various
segments are colored as in Part
a.
The posttranscriptional processing
of yeast tRNATyr