Zinc fingers

Zinc fingersPresented by

Vipin kumar 2014h129007pDiscovered as repeating unit motif later known as zinc finger as gripped and grassped dna.Tf3 of 5s rna gene,repeating structure:a.30aa repeat in the sequence b.Proteolysis results in limit digest of 3kda structuresc.7-11 zn molecules attached

Introduction

Tf3 a structure

Instead of using 2 fold symmetry dna helix,zn finger dna(or rna) binding module linked tendemly in linear fashion.Similar structural feature of ech unit but achieve specific aafinity by varying key aa residues.This modular design allow to design large no.of combination poosilities to recognise sp.sequences.

Unique principle of dna recognitionAA residues involved in interaction

2 antiparallel sheet which contains loop formed by 2 cys,and an helix containing the His-his loopThe two structural units are connected by zn ion.Dna recognition reside mostly in helical region of str. zn fingers are str.independent and linked by flexible linker.

Structure of zn finger

Modular recognition of dna molecule Zif268 TF dna binding domain having 3 zn finger peptide model1aa to 1bp linkages unlike other dna binding motifsEach finger interacts with 3 bp subsite,3 znf motif recognizes 9bp sequenceUses both statrands for recognitonHighly specific in nature

Dna interaction

Specificity of recognition of each zn finger enable to improve specificity to higher level.Main source of specificity -1,3,6 of recognition helix,cross strand interaction at 2 influence specificity hence 4bp overlapping subsites.Selection of specific zn finger by phage display method allow to design zn motif with different specificity.

Zn finger affinity

9bp recognition znf motif linked to designed 18bp recognition zn motif via 2 ways 3*2 or 2*3 znf linkages.Six fingers domain binding 18 bp target sites18 bp sequences offers high specifity,one in whole human genome (4^16=4.2*10^9)3*2F picomolar affinity for dna and more sensitive to mutation allow its preeference over 2*3f construct.Offers single gene recognition tested on 20,000 dna sequence microarray

Improving znf affinity for dna

Fusing poly zn finger motifs with variety of effectors protiens (pabo and collegues,sangamo Inc.)Inhibition of HIV virus expression,zfp targeted to hiv promotor terminal repeat,activated by tat protien represses expressionHuntingtton desease elimination by selectively differentiating mutant extended repeats of CAG nts from wild type(no.of repeats differ) Some application of znf protien

Activation of transcription factor VEGF by zfp invivo in mouse Vegf key regulator and effector in angiogenisis and alternatively spliced.Results in creation of new blood vessels by action on promotor of VEGF gene Splice variants ratio are naturally produced and no leakage og vessels. VEGF stimulation by ZNF protiens Homology directed recombination allow gene editing mediated by znf.Gene targetting by replacing defected gene by hdr.Chimeric zf make ds break and exogenous right dna copy allow gene repalecement by hdr.Uses in gene therapy more efficiently by targetting sp.dna sites to be replaced

Zfp mediated gene correction

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