John O. Thomas

A cytoplasmic chaperonin that catalyzes β-actin folding

Abstract We have isolated a Cytoplasmic chaperonin based on its ability to catalyze the folding of denatured β-actin. The cytoplasmic chaperonin is organized as a multisubunit torold and requires Mg 2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-Independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg 2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that Is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts.

Altered arrangement of the DNA in injection-defective lambda bacteriophage

Abstract We have characterized two variant bacteriophage λ particles, λZ− and λdoc L, that have low infectivity but normal morphology. The low infectivity is due, at least in part, to a defect in DNA injection. This defect is probably the result of an altered location of the right end of the chromosome with respect to the phage tail: the right end of λZ− and λdoc L DNA, in contrast to that of wild-type λ, cannot be cross-linked to the tail. The cross-linking experiments were greatly facilitated by a new technique that allows routine spreading of DNA for electron microscopy without the use of a protein film. We propose that the Z gene product, a tail protein, acts by recognizing a specific feature near the right terminus of the DNA and promoting its insertion into the tail. This feature is presumably missing in most λdoc L particles.

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

Abstract We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and oX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m -Na + or 0·1 m -Na + . An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono- N -ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and oX174 viral DNA with a stoichiometry of one N -ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.

RNA-Helix-Destabilizing Proteins

Publisher Summary This chapter presents a discussion on ribonucleic acid (RNA)-helix-destabilizing proteins. The chapter outlines some aspects of RNA metabolism where the need for helix-destabilizing proteins appears to be evident. The chapter focuses on ribosomal protein S1 from E. coli and protein HD40 from the brine shrimp Artemia salina , a major component of heterogeneous nuclear ribonucleoprotein (hnRNP) particles. In both cases, the in vitro unwinding activity of these proteins can be correlated with their respective functions. The chapter describes the nucleic acid binding and helix-destabilizing properties of protein S1 and HD40. While comparative investigations of analogous proteins from other eukaryotes are needed, HD40 may prove to be a good model for in vitro studies on the structure and function of hnRNP; its physical properties appear to be representative of the glycine-rich core proteins, and it can be purified in relatively large amounts. In contrast to helix-destabilizing proteins participating in dynamic processes that appear to exercise a transient effect on the conformation of the nucleic acid, HD40 is a component of an isolatable nucleoprotein in which the single-stranded RNA is maintained in an unfolded state owing to its interaction with the protein. The chapter recommends the need for in vitro investigations involving purified individual hnRNP proteins and messenger ribonucleic acid (mRNA) precursors along with the development of specific methods for the isolation of native hnRNP.

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native beads-on-a-string structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual beads which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.

Intracellular distribution of a nuclear localization signal binding protein

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.