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Biomedical subjects

P J Butler

Publications and source records attributed to P J Butler.

At least 145 records · Page 8Linked to original sources
PubMed1980

RNA-protein interactions in the assembly of tobacco mosaic virus.

Assembly of tobacco mosaic virus is initiated by the binding of a specific loop of the RNA into the central hole of the disk aggregate of protein subunits. Since the nucleation loop is located about five-sixths along the RNA molecule, subsequent elongation must be bidirectional. We have now measured the rates of elongation in the two directions by determining the lengths of RNA protected from nuclease digestion at different times and using either intact TMV rNA, or RNA with most of the longer tail removed. Comparison of the rates with the protein supplied as either a mixture of disks with A-protein (a mixture of less aggregated states) or just A-protein, shows that different mechanisms and protein aggregates are used for the most rapid growth. When disks are present, they add more rapidly along the longer RNA tail but do not appear to add directly on the shorter tail. In contrast, smaller aggregates (A-protein) can add at both ends of the rod, but do so more slowly. Mechanisms for these processes are discussed. Preliminary results on the binding of the specific hexanucleotide AAGAAG to the disk are given and compared with the known changes on binding nonspecific hexanucleotides or the trinucleotide AAG.

Kinetics↗
PubMed1979

Binding of oligonucleotides to the disk of tobacco-mosaic-virus protein.

The trinucleoside diphosphate A-A-G and the hexanucleotide fraction from a ribonuclease I digest of yeast RNA have been soaked into crystals of the disk aggregate of tobacco mosaic virus protein. At high concentrations these cause disruption of the crystal, probably by mimicking the normal nucleation of assembly. At lower nucleotide concentrations the crystals remain intact and the differences caused by nucleotide binding have been studied by X-ray diffraction. The most obvious change is an upwards movement of about 0.3 nm at the low-radius end of the left radial helix in the protein with some stiffening of the helix so that it now extends visibly in from 4 nm to 6 nm radius. Similar shifts also occur in the right radial and left slewed helices. A positive peak, which is tentatively identified with the bound oligonucleotide, is seen around 4 nm radius and below the right radial helix. The amino acid residues in possible contact with this feature are discussed.

Nucleic Acid Conformation↗
PubMed1979

Location and encapsidation of the coat protein cistron of tobacco mosaic virus. A bidirectional elongation of the nucleoprotein rod.

The coat protein cistron of tobacco mosaic virus has been located on the viral RNA starting between 975 and 1050 nucleotides from the 3'-hydroxyl end. This locates its 5' end close to the origin for virus assembly, where the first protein disk interacts with RNA. It also means that the coat protein mRNA must have a short 5'-untranslated tail and a long (over 500 nucleotides) 3' one. The recovery of characteristic oligonucleotides in nuclease-protected rods during the growth from RNA and a protein disk preparation shows that elongation of the nucleated rods proceeds independently in both directions though, on average, much more rapidly along the longer 5' tail than the shorter 3' tail. Protected RNA of length equal to that in the complete virion is first seen within 6 min, showing that the most rapidly elongated particles are substantially complete by this time.

Base Sequence↗
PubMed1978

Location of tyrosine residues in the disk of tobacco-mosaic-virus protein and comparison of the subunit packing with that of the virus.

The products of iodination of the disk of tobacco mosaic virus coat protein have been investigated in order to locate the reactive amino acid residues in the three-dimensional structure. Reaction occurs mainly with tyrosine-139 and, to a lesser extent, tyrosine-2 and the positions of these modified residues have been determined by X-ray crystallography. Different extents of reaction are found in the two rings of the disk and also, on adding the high salt concentration needed for stabilisation of the crystal during reaction, some conformational changes in the polypeptide chain in the inner part of the disk. Comparison of the relative positions of residues 27 and 139 in the disk and virus shows that little distortion occurs in the outer part of the subunit during the transition between the disk and virus structures.

Amino Acids↗
PubMed1977

Configuration of tobacco mosaic virus, RNA during virus assembly.

When TMV reassembles, the uncoated RNA is folded back along the growing rod, probably down the central hole. This surprising configuration is essential for rapid elongation--presumably supplying RNA to its site of incorporation while keeping the bulk of the free RNA out of the way.

Kinetics↗
PubMed1977

Structure of transcriptionally-active chromatin subunits.

Rat liver chromatin is organized into regions of DNA which differ in degree of susceptibility to attack by the endonucleases DNase I and DNase II. The most nuclease-sensitive portion of chromatin DNA is enriched in transcribed sequences. This fraction may be separated from the bulk of chromatin by virtue of its solubility in solutions containing 2 mM MgCl2. Both transcribed and nontranscribed regions of chromatin are organized into repeating units of DNA and histone, which appear as 100 A beads in the electron microscope. The length of DNA in the repeat unit is the same for these two classes of chromatin (198 +/- 6 base pairs in rat liver); however, the subunits of active, Mg++-soluble chromatin differ from the nucleosomes of inactive regions of chromatin in several respects. Active subunits are enriched in nascent RNA and nonhistone protein and exhibit higher sedimentation values than the corresponding subunits of inactive chromatin.

Animals↗
PubMed1976

Assembly of a spherical plant virus.

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

Capsid↗
PubMed1976

Assembly of tobacco mosaic virus.

The assembly of tobacco mosaic virus requires the presence of a particular protein aggregate, the disk. During the nucleation, a specific region of the RNA interacts with a single disk, to bring about a necessarily cooperative transition from the paired two-layer structure to a short segment of nucleo-protein helix. There is a high selectivity for this region of the TMV RNA, because of the many nucleotides bound at once, and other nucleotide sequences appear only to bind by a different mechanism. Elongation of the nucleated rods can continue with either further disks or the less aggregated 'A-protein' as the protein source, but the continued cooperativity inherent with disks would have some advantages. The rates of the two processes have been separately determined and growth is faster when disks are still present. New experiments show that the breakdown of disks to yield A-protein is relatively slow and it is concluded that virus growth from disks could not proceed through a prior breakdown in solution, but must involve the direct interaction of the disk with the growing nucleoprotein rod. The detailed mechanism of disk addition is not understood but it may involve a directed breakdown, since there is also evidence for the existence of a non-equilibrium form of A-protein which has aggregation kinetics distinct from those of equilibrium A-protein. Some implications for the general assembly pathways of viruses both of the specificity and of the assembly/disassembly cycle during the viral infection are considered.

Kinetics↗