Assembly of a Nucleoprotein Complex Required for DNA Packaging by Bacteriophage
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- 作者：Qin Yang ; Adrienne Hanagan ; and Carlos Enrique Catalano
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：March 11, 1997
- 年：1997
- 卷：36
- 期：10
- 页码：2744 - 2752
- 全文大小：217K
- 年卷期：v.36,no.10(March 11, 1997)
- ISSN：1520-4995
文摘
A critical step in the assembly of bacteriophage 
es/gifchars/lambda.gif" BORDER=0 > is theexcision of a single genome from aconcatemeric DNA precursor and insertion of genomic DNA into an emptyviral capsid. DNA packagingis mediated by the es/gifchars/lambda.gif" BORDER=0 > proteins gpNu1 and gpA, which form an enzymecomplex known as terminase.Initiation of the packaging process requires assembly of theterminase subunits onto cos, the es/gifchars/lambda.gif" BORDER=0 > DNApackaging sequence, and nicking of the duplex, thus forming the12-base-pair "sticky" ends of the maturegenome. We have utilized gel-retardation techniques to examine theinteraction of gpNu1, gpA, andterminase holoenzyme with DNA. Our data demonstrate that gpNu1interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly,the larger gpA subunit binds to DNA,forming two complexes; however, this subunit forms similar complexeswith DNA substrates of randomsequence. All of the nucleoprotein complexes examined aredisrupted by elevated concentrations of NaCland we suggest that altered DNA binding is responsible for the extremesalt sensitivity of the endonucleaseactivity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993)J. Biol. Chem. 268,3056-3065]. DNAbinding by each subunit is strongly affected by the presence of theother, with 10- and 3-fold increasesin the affinity of gpNu1 and gpA, respectively, for DNA. Moreover,our data suggest that the terminasesubunits interact in solution prior to DNA binding. Finally, weprovide evidence that complex I, the firststable intermediate in the packaging pathway, is composed of the matureleft genome end bound to theterminase subunits and demonstrate that dissociation of the complex isquite slow (t1/2 > 8 h).Thesignificance of these data with respect to terminase-mediated genomepackaging is discussed.