David G. Bear

Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore.

The nuclear poly(A)-binding protein, PABPN1, has been previously shown to regulate mRNA poly(A) tail length and to interact with selected proteins involved in mRNA synthesis and trafficking. To further understand the role of PABPN1 in mRNA metabolism, we used cryo-immunoelectron microscopy to determine the fate of PABPN1 at various stages in the assembly and transport of the Chironomus tentans salivary gland Balbiani ring (BR) mRNA ribonucleoprotein (mRNP) complex. PABPN1 is found on BR mRNPs within the nucleoplasm as well as on mRNPs docked at the nuclear pore. Very little PABPN1 is detected on the cytoplasmic side of the nuclear envelope, suggesting that PABPN1 is displaced from mRNPs during or shortly after passage through the nuclear pore. Surprisingly, we also find PABPN1 associated with RNA polymerase II along the chromatin axis of the BR gene. Our results suggest that PABPN1 binds to the polymerase before, at, or shortly after the start of transcription, and that the assembly of PABPN1 onto the poly(A) tail may be coupled to transcription. Furthermore, PABPN1 remains associated with the released BR mRNP until the mRNP is translocated from the nucleus to the cytoplasm.

21 Single-Stranded DNA Binding Proteins

Publisher Summary This chapter focuses on the proteins that bind preferentially and nonspecifically to single-stranded DNA and have no other (enzymatic) activity. These proteins are essential to many physiological functions, including replication, recombination, and repair, in a host of organisms ranging from bacteriophage to higher eukaryotes. Thus, single-stranded DNA binding proteins represent systems that have evolved substantially beyond primitive precursors, which may only have been capable of direct and uncontrolled nucleic acid binding. The chapter describes molecular aspects of the involvement of DNA binding proteins in entire systems of DNA replication, recombination, and repair. Nature and measurement of DNA–protein interactions along with general purification strategies for single-stranded DNA binding proteins are also presented. In addition, the chapter discusses the ways in which the single-stranded DNA binding proteins have been exploited as tools in molecular biological research, particularly in the electron microscopy of biological macromolecules and in certain biochemical assays. All DNA binding proteins seem to operate stoichiometrically (as opposed to catalytically), in that they are present at intracellular levels sufficient to effectively saturate the single-stranded DNA intermediates produced during replication, recombination, and repair. Binding cooperativity is essential in permitting complete coverage of single-stranded sequences and also in effectively destabilizing the small duplex hairpins formed by intrastrand base pairing in single-stranded DNA.

Intranuclear inclusions in oculopharyngeal muscular dystrophy contain poly(A) binding protein 2.

Intranuclear inclusions are one of the ultrastructural hallmarks of oculopharyngeal muscular dystrophy (OPMD), a disorder caused by small polyalanine (GCG) expansions in the gene that codes for a ubiquitous nuclear protein called poly(A) binding protein 2 (PABP2). We studied OPMD skeletal muscle and found that 1.0 to 10.0% of myocyte nuclei contained discreet PABP2 immunoreactive intranuclear inclusions, providing the first direct evidence of the relation between the proposed gene for OPMD and the pathology of OPMD. Ann Neurol 2000;48:812–815

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non‐contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo‐Phe274. Binding of Topo‐Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild‐type topoisomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase–DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase‐mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.

Imaging of metal-coated biological samples by scanning tunneling microscopy.

A method for imaging biological samples by scanning tunneling microscopy (STM) is presented. There are two main difficulties in imaging biological samples by STM: (1) the low conductivity of biological material and (2) finding a method of reliably depositing the sample on a flat conducting surface. The first of these difficulties was solved by coating the samples with a thin film of platinum-carbon. The deposition problem was solved by a method similar to a procedure used to deposit biological molecules onto field ion microscope (FIM) tips. STM images of bacteriophage T7 and filamentous phage fd are shown. The substrate on which the samples were absorbed was atomically flat gold. The images do not show molecular detail due to the metal coating, but the gross dimensions and morphology are correct for each type of virus. Also, the surface density of virus particles increases and decreases in the way expected when the conditions of deposition are changed. These methods allow reliable and reproducible STM imaging of biological samples.

The E. coli rho protein: an ATPase that terminates transcription

Abstract The Rho protein is a toroid-shaped oligomeric ATPase that catalyses thet release of nascent RNA transcripts at discrete sites within the transcription units of E. coli and many coliphages. The signal that specifies the location of the Rho-mediated termination event appears to be quite complex. Multiple sites for Rho-subunit attachment are superimposed on a large region of the RNA transcript that contains little or no secondary structure. RNA polymerase pause sites and template-transcript interactions may also serve to define the actual locations of transcript release.

A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase

We have developed a plasmid expression vector for the study of transcription terminators in Escherichia coli that utilizes the lux genes coding for the enzyme luciferase of the bioluminescent marine bacterium, Vibrio harveyi. The pBR322-derived plasmid, called pHV100, contains the E. coli lac promoter, the polylinker regions from the plasmid vector pUC18, and the V. harveyi lux genes. Insertion of transcription termination sites into the polylinker region results in decreased luciferase expression. Because the bioluminescence genes are not indigenous to E. coli, their expression can be studied in virtually any host strain without the complications of background activity. This facilitates sensitive measurements of terminator efficiency in hosts containing termination factor mutations. Bioluminescence can be easily monitored with high sensitivity, using a rapid photographic technique or a more quantitative photometric assay.

A method for imaging E. coli. RNA polymerase holoenzyme with the scanning tunneling microscope in an aqueous environment

An electodeposition method has been developed for depositing E. coli. RNA polymerase onto a monoatomically flat gold surface. The scanning tunneling microscope has been used to image this enzyme in a high‐humidity or water/glycerol environment, which allows direct observation without metal coating. The deposition technique is reproducible enough to allow optimization of the deposition parameters for controlled electrodeposition. Time‐dependence studies were conducted to help determine the binding mechanism of the protein. The enzyme molecules appear as ordered arrays, amorphous features, or ‘‘jaw‐shaped’’ molecules depending on the deposition conditions used. The ‘‘jaw‐shaped’’ molecules closely resemble those observed by electron microscopy. In some of these a groove 22 A wide can be seen.

Hip Flexion Weakness is Associated with Impaired Mobility in Oculopharyngeal Muscular Dystrophy: A Retrospective Study with Implications for Trial Design

Oculopharyngeal muscular dystrophy (OPMD) is a rare myopathy for which validated outcome measures are lacking, posing a barrier to clinical trials. Our goal was to identify factors associated with impaired mobility in OPMD in order to guide development of surrogate endpoints in future clinical trials. One hundred forty-four individuals with OPMD were included in this retrospective, single-center study. We made novel use of parametric time-to-event analysis to model age at initial use of assistive device for ambulation. We hypothesized that limb weakness and other markers of disease severity are associated with earlier use of assistive devices. 23.6% of individuals (34/144) progressed to use of assistive devices (mean age 66.0 ± 9.6 y). Earlier age at assistive device was associated with hip flexion Medical Research Council grade ≤3 (p <0.0001), earlier disease onset (p <0.0001), and lack of blepharoptosis surgery (p = 0.011). Markers of dysphagia severity were not associated with earlier progression to assistive devices. Our study is the first to show a statistical association between hip flexion weakness and impaired mobility in OPMD, indicating that hip flexion strength could be explored as a surrogate endpoint for use in clinical trials. Since severity of disease features may be discordant within individuals, composite outcome measures are warranted.

Electrodeposition procedure of E. coli RNA polymerase onto gold and deposition of E. coli RNA polymerase onto mica for observation with scanning force microscopy

Molecules of the transcriptional enzyme E. coli RNA polymerase (RNAP) have been deposited using three different deposition methods: (1) passive adsorption onto gold, (2) electrochemical adsorption onto gold and (3) adsorption onto mica. In all cases SFM imaging was straightforward and reliable, and surface coverage by the protein varied with deposition conditions as expected. To determine the nature of the electrochemical treatment on the gold substrate, cyclic voltammetry was performed with various chemical solutions. Finally, a comparison is made between the SFM images of RNAP obtained with these methods and STM images obtained earlier. Both STM and SFM show strikingly similar results; however, heights and widths of individual molecules differ.