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INTEIN MEDIATED PROTEIN SPLICING
Jayanti Tokas1, Rubina Begum1, Shalini Jain2 and Hariom Yadav2
1Department of Biotechnology, JMIT, Radaur
2 NIDDK, National Institute of Health, Bethesda,MD20892, USA
Email: [email protected]
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
PROTEIN SPLICING
Intein Mediated Protein Splicing Occurs Post-translationally
Protein Splicing RNA Splicing
Discovery
In 1990,in Saccharomyces cerevisiae vacuolar ATPaseThe TFP1 gene of S. cerevisiae encodes two proteins. 69kd catalytic subunit of V type ATPase 50kD protein.
73% 77%
N domain Spacer domain C domain
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.
1 284 738 1071
1 273 608
Neurospora vma-1
Yeast TFP1
Restriction fragments of TFP1 were placed under Restriction fragments of TFP1 were placed under an inducible promoter,expressed in E.coli and an inducible promoter,expressed in E.coli and immunoblotted using mAbs to 69kD subunitimmunoblotted using mAbs to 69kD subunit
None of the Abs recognized the spacer domain None of the Abs recognized the spacer domain
N and C domains are joined to form 69kD subunit
ATG stop
XX B E P K N S H Sp
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.
Inteins consisting of only the self splicing domain are
called mini-inteins.
Conserved Motifs
Split inteinsSplit inteins: eg. In DnaE, : eg. In DnaE, the catalytic subunit of the catalytic subunit of DNA polIII in DNA polIII in Nostoc Nostoc PunctiformePunctiforme
A pair of split DnaE genes A pair of split DnaE genes produces two precursor produces two precursor polypeptides.polypeptides.
Transcription and Translation
Trans Protein Splicing
Active DNA Pol cat. subunit
N
N
C
C
dnaE-n dnaE-c
N-intein C-intein
Inactive Precursors
Intein protein splicing domain
(embedded in Myc. leprae gyrase gene)
N
C
Intein protein splicing domain with homing
endonuclease (embedded in S. cerevisiae proton
ATPase)
NC
C
N
Intein protein splicing domain with homing endonuclease--DNA
bound
Endonuclease
454 amino acid(~50kD )protein 454 amino acid(~50kD )protein encoded by the intervening encoded by the intervening sequence of TFP1.sequence of TFP1.
Composed of 2 domains:Composed of 2 domains:
~a splicing domain &~a splicing domain &
~an endonuclease domain.~an endonuclease domain. Domain I comprises the first Domain I comprises the first
182 & last 44 residues.182 & last 44 residues. Compact & globular domain II.Compact & globular domain II.
comprises the183-410 residuescomprises the183-410 residues..
Structure of PI-SceI
VMA intein-Vacuolar Membrane, ATPase intein
An extreme example from a marine cyanobacterium…
RIR is RNR…ribonucleotide reductase
Homing Endonuclease ActivityHoming Endonuclease Activity
Homing is the lateral transfer Homing is the lateral transfer of an intervening sequence to a of an intervening sequence to a homologous allele that lacks homologous allele that lacks the sequence.the sequence.
Catalysed by an endonuclease Catalysed by an endonuclease that recognizes and cleaves the that recognizes and cleaves the target allele.target allele.
Most endonucleases have a Most endonucleases have a conserved DOD conserved DOD sequence(LAGLIDADG)sequence(LAGLIDADG)
Intein mobility (homing)
I-
I+
= extein = intein = homing endonuclease
Homing endonucleases give intervening sequences the ability to be invasive and persistant
I+
I+
Inteins as parasitic genesGene conversion process via double strand break repair
The Mechanism of Protein Splicing
The Mechanism of Protein SplicingStep 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
The ribonucleotide reductase large subunit of the The ribonucleotide reductase large subunit of the Chilo iridescent virusChilo iridescent virus (CIV) contains an intein (CIV) contains an intein having a having a C terminal Glutamine C terminal Glutamine instead of Asn.instead of Asn.
Variations in Splicing Pathway
• 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 (N-terminal) instead of Ser1 or Cys1.
Applications of protein splicing
Rapid purification of target proteins
Temperature sensitive control of Temperature sensitive control of protein protein activity by conditionally activity by conditionally splicing splicing inteins.inteins.
protease
affinity purification
elution
Proteasetreatment
further purification
Affinity purification using a tag
Affinity purification using a modified intein tag
target proteinaffinity tag
target proteinaffinity tag
target proteinaffinity tag
target protein
affinity tag
target protein
Rapid purification of target proteins
Temperature sensitive control of protein activity Temperature sensitive control of protein activity by conditionally splicing inteins.by conditionally splicing inteins.
Temperature sensitive splicing variants of VMA intein can be Temperature sensitive splicing variants of VMA intein can be inserted within transcription factors that can be active only at inserted within transcription factors that can be active only at temperatures at which splicing occurs.temperatures at which splicing occurs.
Gal4AD
Gal4 BDI
UAS
UASUAS
UASTarget Target
TargetTarget
Gal4AD
Gal4AD
Gal4AD
Gal4 BD
Gal4 BDGal4 BD
I
II
Gal80
Gal80
29ºC: No splicing
29ºC: No Splicing
18ºC: Splicing
18ºC: Splicing
(Nature BT, 2004)