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: yadavhariom@gmail.com

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)