Download Requirements for translation re-initiation in Escherichia coli: roles of

Requirements for translation
re-initiation in Escherichia coli:
roles of initiator tRNA and
initiation factors IF2 and IF3
Molecular Microbiology (2008) 67(5), 1012–1026
Jae-Ho Yoo† and Uttam L. RajBhandary*
Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
Presentation by: Liz Gallo
Introduction
Purpose: investigate requirements for translation
re-initiation in E.coli by:
1. constructing a di—cistronic reporter based on
the translationally coupled geneV-geneVII
pair from M13 phage
2. studying the effects of using mutant initiator
tRNA’s
3. studying the effects by modulating IF2 and
IF3 activity
Key Terms
• Di-cistronic – translates 2 proteins
• Polycistronic – translates many proteins
• Intercistronic region – distance between the stop codon of
upstream ORF and the start codon of the downstream ORF
• Reporter – gene that researchers attach to a regulatory
sequence of another gene of interest; often used as an
indication of whether a certain gene has been taken up or
expressed
• Shine-Dalgarno Sequence - is a ribosomal binding site in
the mRNA, generally located 8 base pairs upstream of
the start codon AUG. (consensus sequence)
• M13 phage – circular bacteriophage consisting of ssDNA
• GeneV & Gene VII- genes are coupled in M13 phage
Abbreviations
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CAT – chloramphenicol acetyltransferase
CL – wildtype CAT/fLuc
fLuc – firefly luciferase
am1 – mutant UAG start codon
rbs – SD sequence
What are CAT and fLuc???
• CAT – chloramphenicol
acetyltransferase
– Bacterial enzyme that is
responsible for
chloramphenicol (broad
spectrum antibiotic)
resistance in bacteria
• fLuc – luciferase
– used in bioluminescence,
from the tail of a firefly
– catalyses the
production of light
– Can be measured by:
RLU/OD and immunoblot
assay
Background
• Three pathways of translation initiation
are found in E.coli:
– De novo initiation – 30S ribosomal subunit
binds to mRNA containing a Shine-Dalgarno
(SD) sequence (from scratch)
– Re-initiation (see below)
– Initiation with leaderless mRNA – mRNA
with 0 or very few nucleotides upstream
from start codon
Polycistronic Operons
• Eubacteria contain many genes that are
part of polycistronic operons and appear
to be coupled
• Translational coupling and re-initiation
are important for expression of these
polycistronic operons
Re-initiation
• What is re-initiation?
– Re-initiation occurs when the same
ribosome used to translate an upstream
open reading frame (ORF) also translates a
downstream ORF.
– Couples translation of a downstream gene
to translation of an upstream gene aka
translational coupling
– Little is known about this mechanism
The Study
• To study translational
re-initiation, the
intercistronic region
from gene V - gene VII
from M13
bacteriophage was used
to design and construct
an inducible, dicistronic reporter
system
Di-cistronic reporter
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•
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A 72 nucleotide sequence (last
41-13aa of gene V and first 30 –
10 aa of gene VII and a C inbetween) was linked downstream
of CAT and upstream of fLuc
This represents a di-cistronic
operon – used to confirm that
both reporters (CAT and fLuc)
were co-transcribed and cotranslated
fLuc - servers as a reporter for
translation re-initiation
CAT –monitors de novo initiation
as well as normalizes reinitiation activity to levels of
ribosomes that enter the reinitiation site after translating
CAT
– Under transcriptional control of
inducible arabinose promoter
Creating Di-cistronic Reporter
• Using M13 DNA as a template, a
DNA fragment containing the
entire geneV and part of
geneVII was amplified by PCR
• PCR product was digested with
restriction enzymes, and cloned
• CAT and fLuc also amplified by
PCR cut with restriction enzymes
and ligated to plasmid
• Resulting in: artificial operon
encoding CAT_geneV and
geneVII_fLuc
Di-cistronic reporter system
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Confirms that both reporters
(CAT and fLuc) were cotranscribed and co-translated
E.coli cells were transformed
with WT CL, and used arabinose
as the inducer
Cell extracts were analyzed for
reporter CAT and fLuc activity
and for protein expression level
activity
Assays for fLuc activity showed
an increase in activity with
increasing levels of arabinose
Immunoblot analysis also show
an increase in activity with an
increase of arabinose
How do we know fLuc expression
is coupled to CAT?
• AUG initiation codons of
the CAT and fLUC genes
were mutated
• Mutant initiator tRNA genes
were cloned into reporter
plasmids that contained the
mutant reporter gene with
the corresponding non-AUG
start
– WT = CL, Mutants = subscripts
Results
• When start codon in CAT
was altered there was no
detectable CAT expression
and fLuc expression was
abolished (lane 3)
• When fLuc start codon is
mutated expression of full
length fLuc was abolished
but expression of CAT and
internally initiated
luciferase fragments were
unaffected (lane 4)
Intercistronic Distance
• Increased intercistronic
distance between STOP
codon of CAT and START
codon of fLuc
– SupF – amber (UAG)
supressor
• Re-initiation generally
decreases with increased
intercistronic distance
due to increased
probability of ribosomal
dissociation
Shine-Dalgarno Sequence
• To determine if SD
sequence would
increase translation reinitiation of fLuc a
GAGG sequence was
inserted 9 nucleotides
upstream from the
fLuc start codon
• SD increased fLuc
activity
Activity of mutant initiator
tRNAs in re-initiation
• Changing CAU(wt)
anticodon to CUA and
GAC allowed the
mutant initiator tRNA
to base pair with
mutant start UAG and
GUC codons
Comparison of mutant initiator tRNA U35A36 (CUA)
in de novo initiation of mutant CAT from Cam1L and
re-initiation of mutant fLUC from CLam1
Requirements in initiator tRNA for
translation re-initiation
• Eubacterial initiator
tRNAs are different
from elongator
tRNAs
– Met-tRNAfMet 
fMet-tRNAfMet by
MTF
– Binding of fMettRNAfMet to
ribosomal P site
• Properties include:
– Mismatch at end of
acceptor stem for
recognition by MTF
– 3 consecutive G:C base
pairs in the anticodon
stem for binding to the
ribosomal P-site
tRNA Mutants
• U35A36/G72G73
(G72/G73) –
defective in
formylation
• C30:G40/U35A36
(C30G40) and
U29C30A31:U39G40
A41/U35A36 (3GC)
– defective in
binding of tRNA to
ribosomal Psite
Requirements in initiator tRNA
for translation re-initiation
• E.coli were
transformed with
either Cam1L or
CLam1 reporter
carrying a mutant
initiator tRNA gene
• Extracts assayed
for CAT and fLuc
activity levels
Background Continued
• E.coli express three essential translation initiation
factors that are necessary for efficient and
accurate de novo translation initiation
• Initiation factors, mRNA, fMet-tRNAfMet and the
30S ribosomal subunit form the 30S initiation
complex (IC)
– IF1 – not well studied
– IF2 – facilitates binding of fMet-tRNAfMet to the P site
of the 30S initiation complex (30S IC)
– IF3 – facilitates selection of initiator tRNA and
initiation codon by destabilizing 30S ICs that contain
non-initiator tRNA or non-canonical codon-anticodon
pairing in P site
IF2 activity is important for
efficient re-initiation
• Effects of overproducing
IF2 and MetRS on reinitiation of the mutant
fLuc reporter
– Overproduction
increased the reinitiation efficiency of
U35A36 mutant tRNA
– Overexpression of
MetRS leads to
increases synthesis of
mutant fLuc
Mutant initiator tRNA with higher affinity for
IF2 is more active in re-initiation
• IF2 is required for
efficient reinitiation in vivo and
re-initiation may
have a greater
requirement for IF2
than de novo
initiation
Overexpression of IF3 decreases
efficiency of re-initiation
• E.coli cells
transformed with CL
and expression
plasmids
• Cell extracts were
analyzed using
immunoblots with
anti β-lactamase or
anti-CAT Ab (top) or
anti-CAT and anti
fLuc AB (bottom)
SD sequence and IF3
• Inhibitory effects
of overexpression of
IF3 on re-initiation
were less severe
when a SD sequence
was present
Overexpression of IF3 interferes with
M13 phage reproduction
• Would overproduction of IF3
also reduce the levels of gene
VII protein made in cells
infected with M13 phage?
– E.coli that over produced IF3
were compromised as hosts
for M13
– Overproduction of IF3
interferes with a step
involved with phage
replication/and or assembly,
but not adsorption to the cell
The Big Picture
• Mutant fLuc reporter gene can be
translated by re-initiation from non
AUG codons
• Formylation of aa and tRNA binding to P
site are important for re-initiation
• IF2 is required for re-initiation, and
IF3 also plays a role; overproduction of
IF3 seems to inhibit re-initiation
Model for translation re-initiation in E. coli
PCR Site-Directed
Mutagenesis
• Was used to create tRNA and mRNA mutants
• PCR with olgionucleotide primers that contain the
desired mutation were created. By creating a
mutation during the first cycle in binding the
template DNA strand, a mutation can be introduced.
• After a number of cycles the mutated fragment will
be amplified sufficiently to separate from the
original, unmutated plasmid by a technique such as gel
electrophoresis, and reinstalled in the original
context using standard recombinant molecular biology
techniques.
To Create Mutants – Site Directed
Mutagenesis
• You need two primers, complementary to each other, containing the new
(mutant) sequence flanked by 20 bases on each side. For example,
suppose you have the following sequence in some gene in some plasmid
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CTA CTT CCA GAG ACA ACT GAT CTC TAC TAC TAT GAG CAA TTA AAT GAC AGC GGG
And you want to change it to;
CTA CTT CCA GAG ACA ACT GAT CTC TAC TTC TAT GAG CAA TTA AAT GAC AGC GGG
One primer will be;
. . . . .5' . . CCA GAG ACA ACT GAT CTC TAC TTC TAT GAG CAA TTA AAT GAC AGC 3'
and the other primer will be the exact complement;
. . . . .5' . . GCT GTC ATT TAA TTG CTC ATA GAA GTA GAG ATC AGT TGT CTC TGG 3‘
• Heat the plasmid to separate its strands.
• Anneal mutagenic primers that contain the TTC codon, or its reverse
complement, GAA.
• Perform a few rounds of PCR (about 8) with the mutagenic primers to
amplify the plasmid with the altered codon
Further Study
• Is the 70S ribosome involved in reinitiation?
The END!
Polymerase Chain Reaction
• PCR– method for making many copies of a specific
segment of DNA, starting with very small amounts
(amplifies DNA)
– DNA to be amplified is mixed with DNA oligonucleotides,
Taq polymerase, and primers
– Mix is heated (break H bonds/sep DNA strands) and
cooled (allow DNA primers to anneal)
– Primers hybridize to ends of gene to be amplified and
provide a starting point for Taq P. which synthesizes
complementary strands of DNA
– Go through “thermal cycling” –until enough DNA has been
produced
Transformation
• Add transformation solution (ex CaCl2) to
tube
• Place on ice and add E.coli colony
• Add fragment containing gene(s) of interest
(incubate on ice)
• Heat shock – place tube into a warm heat bath
for a little under a minute, place back on ice
(about 2 minutes)
• Add nutrients (LB nutrient broth) and sit at
room temp (10 minutes)
• Transfer to agar plates (these plates contain
ampicillin selection and mutants require arabinose
inducer)