Download INTEIN MEDIATED PROTEIN SPLICING

INTEIN MEDIATED
PROTEIN SPLICING
Jayanti Tokas1, Rubina Begum1, Shalini Jain2 and Hariom
Yadav2
1Department
2 NIDDK,
of Biotechnology, JMIT, Radaur
National Institute of Health, Bethesda,MD20892, USA
Email: [email protected]
PROTEIN SPLICING
Self
catalyzed
protein
rearrangement
Inactive protein precursor to
biologically active protein
Post translational cellular
process
Intervening sequences INTEIN is
excised out
Flanking
sequences
EXTEINS are
ligated
Altered the “central dogma”
of gene
expression
Intein Mediated Protein Splicing Occurs Post-translationally
Protein Splicing
RNA Splicing
Discovery
In 1990,in Saccharomyces cerevisiae vacuolar ATPase
The TFP1 gene of S. cerevisiae encodes two proteins.
69kd catalytic subunit of V type ATPase
50kD protein.
1
273
608
Neurospora vma-1
73%
77%
Yeast TFP1
1
N domain
284
Spacer domain 738 C domain
1071
Homology maps to N and C terminal regions
TFP1 gene encodes 1071 a.a,119 kD protein which show
homology to catalytic subunit of v type ATPase of neurospora.
Restriction fragments of TFP1 were placed under
an inducible promoter,expressed in E.coli and
immunoblotted using mAbs to 69kD subunit
None of the Abs recognized the spacer domain
N and C domains are joined to form 69kD subunit
X
B
E P
ATG
K
N S
H
Sp
X
stop
X-XbaI,B-BamHI,E-EcoRV,H-HindIII,K-KpnI,N-NacI,P-PstI,S-SacII,Sp-SphI
What are Inteins ???
Genetic elements that disrupt the coding sequence of genes.
Transcribed & translated together with their host protein.
Found in organisms of all 3 domains of life:
Eucaryotes(70 inteins)
Eubacteria (150 inteins)
Archaea(110 inteins)
and in viral and phage proteins.
Uptil now over 350 inteins have been described.
Found in metabolic enzymes, DNA and RNA polymerases,
proteases, ribonucleotide reductases and V-type ATPase.
Inteins consists of two domains.
* Splicing domain which comprises the N and C terminals.
* An endonuclease domain.
Conserved Motifs
Inteins consisting of only the self splicing domain are
called mini-inteins.
Split inteins: eg. In DnaE,
the catalytic subunit of
DNA polIII in Nostoc
Punctiforme
dnaE-n
dnaE-c
Transcription and
Translation
N-intein
A pair of split DnaE genes N
produces two precursor
polypeptides.
C-intein
C
Inactive Precursors
Trans Protein
Splicing
Active DNA Pol cat. subunit
N
C
Endonuclease
C
C
N
N
C
N
Intein protein
splicing domain
(embedded in Myc. leprae
gyrase gene)
Intein protein splicing
domain with homing
endonuclease
(embedded in S. cerevisiae proton
ATPase)
Intein protein splicing
domain with homing
endonuclease--DNA
bound
Structure of PI-SceI

454 amino acid(~50kD )protein
encoded by the intervening
sequence of TFP1.
 Composed of 2 domains:
~a splicing domain &
~an endonuclease domain.
 Domain I comprises the first
182 & last 44 residues.
 Compact & globular domain II.
comprises the183-410 residues.
VMA intein-Vacuolar
Membrane, ATPase intein
An extreme example from a marine cyanobacterium…
RIR is RNR…ribonucleotide reductase
Homing Endonuclease Activity

Homing is the lateral transfer
of an intervening sequence to a
homologous allele that lacks
the sequence.
 Catalysed by an endonuclease
that recognizes and cleaves the
target allele.
 Most endonucleases have a
conserved
DOD
sequence(LAGLIDADG)
Intein mobility (homing)
Inteins as parasitic genes
Gene conversion process via double strand break repair
I+
I+
I-
I+
= extein
= intein
= homing endonuclease
Homing endonucleases give intervening sequences the ability to be
invasive and persistant
The Mechanism of Protein Splicing
The Mechanism of Protein Splicing
Step 1: Formation of an ester intermediate by an N-O acyl
rearrangement of the conserved Ser /Cys residue at the
upstream splice junction.
Step 2: Formation of a branched intermediate by transesterification
Step 3: Intein excision by peptide bond cleavage coupled to
succinimide formation involving the conserved Asn residue
at the downstream splice junction.
Step 4: Spontaneous O-N acyl rearrangement in formation of peptide
bond between 2 exteins.
The Mechanism of Protein Splicing
Step 1
Acyl N/O shift (or N/S)
The Mechanism of Protein Splicing
Step 2 Transesterification
The Mechanism of Protein Splicing
Step 3 Asn cyclization/succinamide formation
The Mechanism of Protein Splicing
Step 4 Acyl O/N shift (or S/N) & Succinimide hydrolysis
The Mechanism of Protein Splicing
Variations in Splicing Pathway

The ribonucleotide reductase large subunit of the
Chilo iridescent virus (CIV) contains an intein
having a C terminal Glutamine instead of Asn.
• An unique RNR intein from Carboxydothermus
hydrogenoformans(Chy) has a C terminal Aspartate.
• K1bA
inteins,
Mycobacterial
DnaB
inteins and Deinococcus
radiodurans Snf2 inteins
begin with Alanine (Nterminal) instead of Ser1 or
Cys1.
Applications of protein splicing
 Rapid purification of target proteins
 Temperature sensitive control of
protein activity by conditionally
splicing inteins.
 Rapid purification of target proteins
Affinity purification
using a tag
affinity tag
target protein
affinity purification
affinity tag
target protein
elution
affinity tag
target protein
Protease
treatment
affinity tag
protease
target protein
further purification
target protein
Affinity purification using a modified
intein tag
 Temperature
sensitive control of protein activity
by conditionally splicing inteins.
Temperature sensitive splicing variants of VMA intein can be
inserted within transcription factors that can be active only at
temperatures at which splicing occurs.
I
Gal4 BD
I
Gal4
AD
29ºC: No splicing
18ºC: Splicing
Gal4
AD
Gal4 BD
UAS
UAS
Gal80
I
29ºC: No Splicing
Gal4
AD
Gal4 BD
UAS
I
Gal4 BD
Gal80
Gal4
AD
UAS
18ºC: Splicing
(Nature BT, 2004)