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måndag 17 november 2014

Vuonna 2007 osoitettiin VP30 proteiinin sitoutuvan Ebov mRNA:han tehostamaan transkriptiota

Mikä alue VP30 proteiinissa tekee interaktion mRNA:n kanssa?

 Avainaminohapot, jotka tukevat RNA-sitomisaktiivisuutta ovat VP30 proteiinin  aminohappotähteissä 26-40, arginiinipitoisessa alueessa. .

John SP, Wang T, Steffen S, Longhi S, Schmaljohn CS, Jonsson CB.
J Virol. 2007 Sep;81(17):8967-76. Epub 2007 Jun 13.

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L.
VP30 acts in trans with an RNA secondary structure 
 upstream of the first transcriptional start site to modulate transcription.

 Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to ..90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays.

VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn(2+), but not Mg(2+), enhanced activity.
Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region.

VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences.
To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

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