INTEIN MEDIATED PROTEIN SPLICING

1. 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

2. 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

3. Intein Mediated Protein Splicing Occurs Post-translationally

4. Protein Splicing RNA Splicing

5. Discovery In 1990,in Saccharomycescerevisiae vacuolar ATPase The 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. 1 273 608 Neurospora vma-1 Yeast TFP1 1 738 284 1071

6. 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 N S X B E P K H Sp X ATG stop X-XbaI,B-BamHI,E-EcoRV,H-HindIII,K-KpnI,N-NacI,P-PstI,S-SacII,Sp-SphI

7. 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.

8. 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.

9. Split inteins: eg. In DnaE, the catalytic subunit of DNA polIII in Nostoc Punctiforme A pair of split DnaE genes produces two precursor polypeptides. dnaE-n dnaE-c Transcription and Translation N-intein C-intein N C Inactive Precursors Trans Protein Splicing Active DNA Pol cat. subunit N C

10. C N C N C N Endonuclease Intein protein splicing domain with homing endonuclease--DNA bound Intein protein splicing domain (embedded in Myc. leprae gyrase gene) Intein protein splicing domain with homing endonuclease (embedded in S. cerevisiae proton ATPase)

11. 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

12. An extreme example from a marine cyanobacterium… RIR is RNR…ribonucleotide reductase

13. 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)

14. I+ I- I+ Intein mobility (homing) Inteins as parasitic genes Gene conversion process via double strand break repair I+ = intein = homing endonuclease = extein Homing endonucleases give intervening sequences the ability to be invasive and persistant

16. The Mechanism of Protein Splicing

17. 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.

18. The Mechanism of Protein Splicing Step 1 Acyl N/O shift (or N/S)

19. The Mechanism of Protein Splicing Step 2 Transesterification

20. The Mechanism of Protein Splicing Step 3 Asn cyclization/succinamide formation

21. The Mechanism of Protein Splicing Step 4 Acyl O/N shift (or S/N) & Succinimide hydrolysis

22. The Mechanism of Protein Splicing

23. 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.

24. An unique RNR intein from Carboxydothermus hydrogenoformans(Chy) has a C terminal Aspartate.

25. K1bA inteins, Mycobacterial DnaB inteins and Deinococcus • radiodurans Snf2 inteins begin with Alanine(N-terminal) instead of Ser1 or Cys1.

26. Applications of protein splicing • Rapid purification of target proteins • Temperature sensitive control of protein activity by conditionally splicing inteins.

27. Rapid purification of target proteins Affinity purification using a tag Affinity purification using a modified intein tag affinity tag target protein affinity purification target protein affinity tag elution target protein affinity tag Protease treatment protease affinity tag target protein further purification target protein

28. 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 AD 29ºC: No splicing 18ºC: Splicing Gal4 AD Gal4 BD I Gal4 BD Target Target UAS UAS Gal80 29ºC: No Splicing I I Gal80 Gal4 AD Gal4 AD Gal4 BD Gal4 BD Target Target UAS UAS 18ºC: Splicing (Nature BT, 2004)

30. Thank you