From these studies, two similar models of the coat protein subunit were obtained for each state. Recently, two sets of subnanometer models based on cryo-electron microscopy data for P22's procapsid and mature capsid were reported ( 9, 12).
In the case of bacteriophage P22, in the absence of scaffolding protein, coat protein gives rise to aberrant structures, including spirals, which result from a misplacement of pentons and a change in the curvature of the capsid subunits, and petite T=4 capsids ( 2, 4). These proteins, often called scaffolding proteins (SPs), are necessary to initiate assembly and direct the correct morphology of viral capsids. In addition, many double-stranded DNA (dsDNA) viruses, including herpesviruses, and the tailed bacteriophages T4, P2, λ, ϕ29, and P22 use auxiliary proteins to assist coat protein assembly ( 3). According to Caspar and Klug's icosahedral virus symmetry theory, coat proteins must adopt quasiequivalent conformations in order to generate a capsid ( 1). Icosahedral viral capsids are commonly formed by multiple copies of a single capsid protein (coat protein ), organized as hexameric and pentameric capsomers. Viruses have a limited genome capacity, which results in a minimal number of distinct proteins used to build it. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro.
#SECONDARY STRUCTURE PROTEIN SCAFFOLD SERIES#
In the case of yeast telomerase, we propose that the RNA serves a very different function, providing a flexible tether for the protein subunits.Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In the best-studied ribonucleoprotein enzyme, the ribosome, the RNAs have specific three-dimensional structures that orient the functional elements. Deletion mutagenesis provides evidence that the Sm arm exists in vivo and can be shortened by 42 predicted base pairs with retention of function therefore, precise positioning of Sm proteins, like Est1p, is not required within telomerase.
These arms emanate from a central catalytic core that contains the template and Est2p-binding region. The model for TLC1 has three long quasihelical arms that bind the Ku, Est1p, and Sm proteins. We present the Est1p relocation experiment in the context of a working model for the secondary structure of the entire TLC1 RNA, based on thermodynamic considerations and comparative analysis of sequences from four species. We now show that the Est1p-binding domain of the RNA can be moved to three distant locations with retention of telomerase function in vivo. In particular, a bulged stem structure binds the essential regulatory subunit Est1p. In the yeast Saccharomyces cerevisiae, distinct regions of the 1.2-kb telomerase RNA (TLC1) bind to the catalytic subunit Est2p and to accessory proteins.