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E Wahle

Publications and source records attributed to E Wahle.

At least 37 records · Page 2Linked to original sources
PubMed1994

Immunodetection of poly(A) binding protein II in the cell nucleus.

During the polyadenylation of pre-mRNA in vitro, poly(A) binding protein II (PAB II) binds to the growing poly(A) tail, stimulating its extension. The subcellular localization of PAB II was investigated with an antibody affinity-purified from rabbit serum raised against the purified protein. Immunofluorescence microscopy detected PAB II exclusively in the cell nucleus, both in a widespread staining and in more intensely stained "speckles." PAB II was excluded from the nucleoli. By electron microscopy, PAB II was also found almost exclusively in the nucleus, predominantly in clusters of interchromatin granules, likely corresponding to the speckles observed by immunofluorescence microscopy, and in perichromatin fibrils, which represent nascent transcripts and probably the sites of pre-mRNA processing. In addition, electron microscopy also detected PAB II in nucleoli. The distribution corresponds largely to that of other factors involved in the processing of pre-mRNA and is thus in agreement with the proposed role of the protein in polyadenylation.

Blotting, Western↗
PubMed1993

Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides.

The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different fractions of the same preparation. Whereas PAB II stimulated poly(A) polymerase, PAB I was an inhibitor. In analytical ultracentrifugation, the predominant form of PAB II was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a distinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The dependence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KD of 2 x 10(-9) M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retardation experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.

Animals↗
PubMed1993

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.

Adenosine Triphosphate↗
PubMed1992

The end of the message: 3'-end processing leading to polyadenylated messenger RNA.

Almost all messenger RNAs carry a polyadenylate tail that is added in a post-transcriptional reaction. In the nuclei of animal cells, the 3'-end of the RNA is formed by endonucleolytic cleavage of the primary transcript at the site of poly(A) addition, followed by the polymerisation of the tail. The reaction depends on specific RNA sequences upstream as well as downstream of the polyadenylation site. Cleavage and polyadenylation can be uncoupled in vitro. Polyadenylation is carried out by poly(A) polymerase with the aid of a specificity factor that binds the polyadenylation signal AAUAAA. Several additional factors are required for the initial cleavage. A newly discovered poly(A)-binding protein stimulates poly(A) tail synthesis and may be involved in the control of tail length. Polyadenylation reactions different from this scheme, either in other organisms or under special physiological circumstances, are discussed.

Animals↗
PubMed1992

Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.

Animals↗
PubMed1991

Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors.

Polyadenylation of messenger RNA precursors requires the nucleotide sequence AAUAAA and two factors: poly(A) polymerase and a specificity factor termed cleavage and polyadenylation factor (CPF). We have purified CPF from calf thymus and from HeLa cells to near homogeneity. Four polypeptides with molecular masses of 160, 100, 73, and 30 kDa cofractionate with CPF activity. Glycerol gradient centrifugation and gel filtration indicate that these four proteins form one large complex with a sedimentation constant of 12 S, a Stokes radius near 100 A, and a native molecular mass near 500 kDa. Purified CPF binds specifically to an RNA that contains the AAUAAA sequence. Mutation of the AAUAAA sequence inhibits CPF binding as well as polyadenylation. Purified CPF contains only trace amounts of RNA and does not react with antibodies against common epitopes of small nuclear ribonucleoprotein particles. Thus, contrary to previous indications, CPF does not appear to be a small nuclear ribonucleoprotein particle.

Animals↗
PubMed1991

A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on a specificity factor, CPF, recognizing the polyadenylation signal AAUAAA. This paper describes an apparently novel poly(A)-binding protein that acts as a second specificity factor, mediating the recognition of the growing poly(A) tail. A transition from a slow initiation phase of polyadenylation to rapid elongation occurs when the growing tail is long enough to serve as a binding site for the poly(A)-binding protein. Elongation of an RNA carrying a tail of 10 or more adenylate residues can occur independently of CPF. A sharp decrease in the poly(A) chain growth rate after the addition of approximately 200 adenylate residues invites speculations about a role of the poly(A)-binding protein in poly(A) tail length control.

Animals↗
PubMed1991

Purification and characterization of poly(A) polymerase from Saccharomyces cerevisiae.

Poly(A) polymerase was purified 22,000-fold to homogeneity from a whole cell extract of Saccharomyces cerevisiae with a yield of 22%. The enzyme is a monomeric polypeptide with a denatured molecular weight of 63,000. Incorporation of labeled ATP into acid-precipitable material by the purified enzyme proceeds faster with manganese than with magnesium ions. Various RNA homopolymers as well as Escherichia coli tRNA or rRNA can serve as primers. An RNA that terminates at the natural poly(A) site of the CYC1 gene is not more efficiently elongated than several nonspecific substrates, indicating the requirement for additional factors to provide specificity. Elongation of the primer is distributive. Covering of a poly(A) primer with poly(A)-binding protein reduces the enzyme's activity more than 10-fold.

Carrier Proteins↗
PubMed1991

Purification and characterization of a mammalian polyadenylate polymerase involved in the 3' end processing of messenger RNA precursors.

A polyadenylate polymerase involved in the polyadenylation of pre-mRNA has been purified 6,000-fold to apparent homogeneity from extracts of calf thymus. In the last purification step, anion exchange chromatography separates the enzyme into three major peaks that are indistinguishable by other physical or functional criteria. On denaturing polyacrylamide gels, the two predominant forms of poly(A) polymerase have molecular weights of 57,000 and 60,000. In solution, the enzyme is a monomer. It polymerizes exclusively ATP. The reaction is distributive and proceeds linearly without any lag phase. The requirement for a primer can be satisfied by any of a number of polyribonucleotides. A significantly higher activity in the presence of Mn2+ as opposed to Mg2+ is due to a hundredfold higher affinity for the primer terminus. In the presence of mg2+ and of a specificity factor partially purified from HeLa cells, the enzyme specifically polyadenylates an RNA that ends at the natural adenovirus L3 polyadenylation site. This reaction depends on the AAUAAA polyadenylation signal.

Animals↗
PubMed1991

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.

Amino Acid Sequence↗
PubMed1989

The dnaB-dnaC replication protein complex of Escherichia coli. I. Formation and properties.

The complex formed between the dnaB and dnaC replication proteins of Escherichia coli is stabilized by ATP binding to dnaC. The dnaB6-dnaC6-ATP6 complex can be maintained without ATP hydrolysis at a concentration as low as 5 x 10(-10) M. The complex is also formed with adenosine 5'-(gamma-thio)triphosphate but generates little or no dnaB activity, suggesting a requirement for ATP hydrolysis in the subsequent stage of binding of the complex to DNA. In this step, dnaC is released, leaving dnaB to function on the associated DNA.

Adenosine Triphosphate↗
PubMed1989

The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions.

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.

Adenosine Triphosphatases↗
PubMed1988

The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase.

A protein in extracts of Escherichia coli that specifically binds the stabilizing par sequence of pSC101 was identified as DNA gyrase. The purified enzyme protects par against digestion by DNase I and exonuclease III. Competition assays demonstrate that gyrase has a 40-fold higher affinity for the 100-bp par sequence than for nonspecific DNA and that par is the major gyrase-binding site in pSC101 derivatives used in this and other studies. Within par, AT-rich sequences occur with a pronounced 10-bp periodicity that is shifted by 5 bp from a similar periodicity of GC-rich sequences. As judged by DNase I digestion, the GC sequences are exposed on the outside of the DNA wrapped around gyrase. The data suggest that the site-specificity of DNA gyrase may be partly determined by the bendability of the DNA. A 4-bp deletion that interferes with Par function in vivo also reduces the affinity for gyrase in vitro. However, a deletion of par causes little reduction in superhelical density in vivo. We conclude that DNA gyrase, while involved in the Par function, may not affect plasmid stability through its supercoiling activity or by an influence on DNA replication.

Bacterial Proteins↗
PubMed1985

Effect of DNA gyrase inactivation on RNA synthesis in Escherichia coli.

The average chain growth rates of rRNA and of total RNA were not affected by a thermal inactivation of DNA gyrase in a temperature-sensitive gyrB mutant of Escherichia coli. The fact that total RNA synthesis decreased under these conditions suggests that transcription is primarily affected at the step of chain initiation. The fraction of rRNA in total pulse-labeled RNA was not altered by inactivation of the enzyme, indicating that the latter is not required to actively maintain a high rate of synthesis of this RNA species.

Escherichia coli↗