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P L Wollenzien

Publications and source records attributed to P L Wollenzien.

14 recordsLinked to original sources
PubMed1989

Structures of human and rabbit beta-globin precursor messenger RNAs in solution.

The structures in solution of human and rabbit beta-globin precursor messenger RNAs containing their first intervening sequence have been investigated. This was accomplished by chemical probing experiments to determine sites of potential base pairing, and by cross-linking experiments to determine the sites of long-range interactions. Secondary structures for both molecules were predicted by using this information. Both molecules are arranged into two separate domains. The first domain, consisting of the first exon, contains several long-range interactions between the beginning of the molecule and sites adjacent to the donor splice site and a partially conserved stem/loop structure. The second domain contains part of the intervening sequence and the beginning of the second exon. The secondary structures involved in the second domain are different in the two molecules. These studies indicate a lack of connection between the donor and acceptor splice sites in these two molecules on the level of the secondary structure. Furthermore, given the absence of strongly conserved structures, it is unlikely that there could be any strict requirements for secondary structures that influence splice site usage.

Animals↗
PubMed1987

The secondary structure of a messenger RNA precursor probed with psoralen is melted in an in vitro splicing reaction.

The secondary structure of the SP6/mouse insulin precursor RNA was determined by psoralen cross-linking experiments. A series of long-range contacts occur within the left half of the pre-mRNA that contains the intervening sequence. Multiple secondary structures for the pre-mRNA exist since some of the interactions share common sites. In splicing buffer but without the splicing extract added, many of these interactions are stable up to at least 50 degrees C. These interactions, however, are dissociated during the in vitro splicing reaction. This dissociation requires ATP and it occurs during the first 30 min. of the splicing reaction. Pre-mRNAs containing psoralen cross-links in different locations within the RNA molecule were purified and used as substrates for in vitro splicing. Psoralen cross-links at any of the double-stranded regions resulted in complete inhibition of the splicing reaction. This indicates that destabilization of the secondary structure of the SP6/mouse insulin pre-mRNA is necessary for in vitro splicing.

Animals↗
PubMed1987

Inhibition of in vitro splicing of a mouse insulin pre-mRNA by covalent cross-links in the intron region.

Recent studies have indicated that in vitro splicing of a mouse insulin pre-mRNA by a HeLa cell nuclear extract is accompanied by the unwinding of substrate RNA. The present experiments were performed to determine whether this melting of the secondary structure of the precursor RNA is essential for the splicing reaction. 32P-labelled mouse insulin pre-mRNA synthesized in vitro in a SP6 transcription system was cross-linked with aminomethyltrimethyl psoralen and fractionated by polyacrylamide gel electrophoresis. RNA species containing different intramolecular cross-links were eluted from the gel and the sites of cross-links were mapped by primer extension analysis using synthetic oligonucleotide primers. Under conditions that allow accurate in vitro splicing of intact pre-mRNA, precursor molecules with psoralen cross-links within their intron region were not spliced by a HeLa cell nuclear extract. This observation strongly supports the assumption that unwinding of precursor RNA molecules is necessary for the splicing reaction.

Animals↗
PubMed1985

Three-dimensional arrangement of the Escherichia coli 16 S ribosomal RNA.

A model for the arrangement of the Escherichia coli 16 S ribosomal RNA in the 30 S ribosomal subunit is given. This model is based on the 16 S ribosomal RNA secondary structure, intramolecular RNA crosslinking results, protein-RNA interactions, and the locations of proteins within the 30 S subunit. These considerations allow placement of most of the RNA helices in approximate positions. The overall shape (that of an asymmetric Y) is very reminiscent of the description of the shape of the RNA made by direct determinations and is reasonably correlated to the appearance of the 30 S subunit. The identities of the three major secondary-structure domains of the 16 S ribosomal RNA are, for the most part, preserved. In addition, many close contacts between the 5' and middle RNA domains occur in the body of the particle. The 3'-terminal domain is situated in the central part of the model. This position corresponds to the region between the head and the platform structure in the 30 S subunit. The regions that represent the general locations of the messenger RNA and transfer RNA binding sites can be identified in the model.

Cross-Linking Reagents↗
PubMed1985

Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy.

Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.

Cross-Linking Reagents↗
PubMed1983

RNA splicing in neurospora mitochondria: structure of the unspliced 35S precursor ribosomal RNA detected by psoralen cross-linking.

The structure of the unspliced 35S precursor rRNA of Neurospora mitochondria was studied by psoralen photochemical cross-linking. The results show that when the 35S RNA is cross-linked in ribonucleoprotein particles (RNPs) under appropriate conditions, the predominant configuration is a 2.2 kb intron loop which brings opposite splice sites into proximity; that the predominant secondary structural feature in the free RNA is a relatively large hairpin (length = 0.105 kb) in the center of the molecule at or near the 5' splice site; that the intron loop and the central hairpin are different configurations of sequences at or near the 5' splice site; and that the intron loop is stabilized by protein components of RNPs. Based on the structures detected by psoralen photochemical cross-linking, we propose a mechanism for the splicing of the Neurospora mitochondrial precursor rRNA. We propose further that certain features of this mechanism may be relevant to the splicing of other RNAs, including eucaryotic mRNAs.

Base Sequence↗
PubMed1983

Use of psoralen monoadducts to compare the structure of 16S rRNA in active and inactive 30S ribosomal subunits.

E. coli 30S ribosomes in the inactive conformation were irradiated at 390 nm in the presence of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT). This produces monoadducts in which AMT is attached to only one strand of an RNA duplex region. After unbound AMT was removed, some ribosomes were activated and then subjected to 360 nm irradiation; others were reirradiated without activation. Electron microscopic examination of 16S rRNA extracted from these two samples showed covalent rRNA loops indicative of rRNA crosslinks. The general pattern of loops closely matched that seen previously after direct psoralen crosslinking of 30S particles. However, the frequency of occurrence of one major class of loops formed by crosslinks between residues near position 500 and the 3' end was substantially lower for the activated samples, implying that the structure of the 16S rRNA in active and inactive 30S particles is different.

Cross-Linking Reagents↗
PubMed1982

Marking the polarity of RNA molecules for electron microscopy by covalent attachment of psoralen-DNA restriction fragments.

We have developed a general procedure for covalently attaching complementary restriction fragments to RNA molecules and visualizing the fragment in the electron microscope after denaturation. This is accomplished by creating psoralen monoadducts on the restriction fragment by irradiation at 390 nm before hybridization to the RNA and subsequently converting the monoadducts to RNA . DNA crosslinks by irradiation at 360 nm. Using this procedure, we have determined the location of a crosslink placed in Escherichia coli 165 rRNA by a previous irradiation. A consistent assignment is obtained by marking the crosslinked RNA with restriction fragments complementary to the 5' end or the 3' end of the 165 rRNA molecule.

DNA↗
PubMed1980

Structure and topology of 16S ribosomal RNA. An analysis of the pattern of psoralen crosslinking.

All of the eleven psoralen crosslinking features previously mapped by electron microscopy of the 16S rRNA can be matched by energetically stable duplex regions found by a computer search of the sequence. One additional particularly stable duplex found in the sequence has subsequently been seen in the microscope. There is no indication that a best or unique fit of the sequence to the crosslinking data has been found. However, it is encouraging that all twelve assignments involve non-overlapping regions of the sequence which allows their simultaneous construction. The resulting general pattern of secondary structure is very different from previous suggestions for rRNAs or other complex RNAs. It is an RNA chain folded in space much like a typical protein chain. Because of the interwound nature of double helices, base pairing between distant regions of the sequence might result in topologically knotted structures. However, examination of available electron microscopic data suggests that the 16S rRNA does not contain any knots.

Base Sequence↗
PubMed1978

Structure of psoralen-crosslinked ribosomal RNA from Drosophila melanogaster.

Ribosomal RNA from Drosophila melanogaster photoreacted with hydroxymethyltrioxsalen has been examined by electron microscopy. Reproducible patterns of hairpins were found in both the 26S and 18S RNA. The frequency of these hairpins and the amount of incorporated drug were dependent upon the conditions under which the crosslinking was performed. A prominent central hairpin occurs in the 26S RNA and the break that interrupts the continuity of the RNA chain is located within it. In addition to several small hairpins, the crosslinked 18S RNA contains a large open loop.

Animals↗