Pancreatic Ribonuclease Digestion Research Articles

Domains on the hepatitis C virus internal ribosome entry site for 40s subunit binding.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

The initiator transfer ribonucleic acid from yellow lupin seeds, correction of nucleotide sequence and crystallization

Two methionine specific tRNAs from yellow lupin seeds have been purified to homogeneity. Initiator tRNA (tRNA i Met) but not tRNA m Met was charged with Escherichia coli methionyl-tRNA synthetase. The nucleotide composition, T i and pancreatic RNase digestion fingerprints and nucleotide sequence of lupin tRNA i Met showed its identity with wheat germ and bean initiator tRNAs. The differences in the primary structure of the lupin tRNA i Met observed by other authors have not been confirmed. We have defined the conditions under which single crystals of lupin tRNA i Met can be grown reproducibly.

The nature of the interaction of eukaryotic initiation factor 2 with double-stranded RNA.

In addition to binding messenger RNA molecules at specific sequences, eukaryotic initiation factor 2 (eIF-2) also binds to double-stranded RNA (dsRNA). The dsRNA is a powerful inhibitor of initiation of eukaryotic translation, causing the inactivation of eIF-2, but in the presence of certain mRNA templates, dsRNA fails to establish inhibition. Such mRNA templates bind to eIF-2 with higher affinity than does dsRNA, while globin mRNA, a template sensitive to inhibition, binds with lower affinity. Here, the nature of the interaction between dsRNA and eIF-2 was studied by examining both the binding of eIF-2 to Penicillium chrysogenum dsRNA molecules carrying 32P label at their 5' ends, and the ability of eIF-2 to protect such label against pancreatic ribonuclease digestion. The results reveal binding sites for eIF-2 at the 5' ends, as well as throughout internal regions of the dsRNA molecule. At least 15 molecules of eIF-2 can be accommodated on a 3000-base molecule of P. chrysogenum dsRNA. eIF-2 protects a 105-base-pair 5'-terminal fragment in dsRNA against digestion, but exhibits no noticeable preference for the 5' ends. By contrast, eIF-2 fails to protect label at the 5' ends of denatured dsRNA molecules, even though it binds to them at internal sites more avidly than to native dsRNA. Binding of eIF-2 to dsRNA is not restricted to specific sequences: eIF-2 binds with equal affinity to the synthetic dsRNA sequence, poly(rI . rC). The data support the interpretation that eIF-2 recognizes the A conformation in dsRNA rather than sequence. Apparently binding of eIF-2 at sites spaced 200 base pairs apart prevents relaxation of the intervening length of the double helix, thereby stabilizing the dsRNA molecule against ribonuclease attack. These results show that, even though dsRNA and mRNA compete in their binding to eIF-2, the structural features recognized by eIF-2 in these RNA species are distinct.

Hepatitis B virus particles of plasma and liver contain viral DNA-RNA hybrid molecules

Hepatitis B virions in plasma (Dane particles) are known to contain small circular DNA molecules. The experiments described here indicate that virions in plasma, as well as particles from hepatitis B virus-infected human liver, also contain viral DNA-RNA hybrid molecules, and deoxynucleotides can be incorporated into the DNA of these hybrids by DNA polymerase activities in the virions. Thus, two viral DNA synthetic reactions appear to take place in virions: repair of the single-stranded region of circular DNA molecules and synthesis or elongation of the DNA strand of DNA-RNA hybrid molecules. Centrifugation of virion nucleic acid to equilibrium in Cs 2SO 4 density gradients revealed the presence of viral DNA-RNA hybrid molecules over a density range of 1.45 to 1.60 g/cm 3. Distinct species of hybrid molecules were found with an average density of 1.57 g/cm 3 in Dane particles and 1.52 and 1.57 g/cm 3 in particles from liver. Fractionation of nucleic acid from Cs 2SO 4 density gradients by gel electrophoresis demonstrated that the majority of hybrid molecules migrated faster than molecules with the density of pure DNA (1.42 g/cm 3). One notable exception was the finding of DNA-RNA hybrid molecules migrating slower than open circular viral DNA. Characterization of viral DNA-RNA hybrids by heat denaturation Cs 2SO 4 density gradient fractionation, and recombinant ] single-stranded probe hybridization revealed that the hybrid molecules consisted of viral plus-strand RNA hydrogen bonded to viral minus-strand DNA sequences. Data obtained by pancreatic ribonuclease digestion revealed that the hybrid molecules at density 1.45 to 1.52 g/cm 3 contained HBV RNA strands base paired over only part of their length in contrast to the hybrid species at density 1.57 g/cm 3 which contained RNA strands apparently base paired over most of their length. Further characterization showed that the hyrid at 1.57 g/cm 3 contained genome-length minus-strand viral DNA. The experiments rule out the possibility that the hybrid molecules are transcriptional complexes. Data presented in a companion manuscript indicate that the hybrid molecules may represent intermediates in the synthesis of viral DNA in the endogenous DNA polymerase reaction.

Ribosomal protein L16 binds to the 3′-end of transfer RNA

Escherichia coli 50 S ribosomal subunits were reconstituted with and without protein L16 present. The latter particles, although active in puromycin reaction, were unable to use CACCA-Phe as an acceptor substrate. We also found that L16 interacts directly with this oligonucleotide and, in the complex with tRNA, protects its 3′-end from pancreatic ribonuclease digestion. We suggest that the role of L16 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its A-site.

Effect of 2'-O-methylation on the structure of mammalian 5.8S rRNAs and the 5.8S-28S rRNA junction.

The mammalian 5.8S rRNA contains a partially 2'-O-methylated uridylic acid residue at position 14 which is largely or entirely methylated in the cytoplasm (Nazar, R.N., Sitz, T.O. and Sommers, K.D. (1980) J. Mol. Biol. 142, 117-121). The effect of this methylation on the 5.8S RNA structure and 5.8-28S rRNA junction was investigated using both chemical and physical approaches. Electrophoretic studies indicated that the free 5.8S rRNA can take on at least two different conformations and that the 2'-O-methylation at U14 restricts the molecule to the more hydrodynamically open form. Structural studies using limited pancreatic or T1 ribonuclease digestion indicated that the methylated conformation was more susceptible to digestion, consistent with a more open tertiary structure. Modification-exclusion studies indicated that the first 29 nucleotides at the 5' end and residues 140 through 158 at the 3' end affect the 5.8S-28S rRNA interaction, supporting previous suggestions that the 5.8S RNA interacts with its cognate high molecular weight component through its termini. These results also suggested that the 2'-O-methylated uridylic acid residue plays a role in the 5.8S-28S rRNA interaction and thermal denaturation studies confirmed this by showing that methylation destabilizes the 5.8S-28S rRNA junction. The 5.8-28S rRNA interaction appears to be more complex than previously believed.

Characterization of the secondary structure features of Escherichia coli, Caldariella acidophila and mammalian ribosomal RNA species by chemical modification of sterically exposed bases.

The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G . U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile, nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 degrees C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkali-stable, derivatives with 1 M methoxyamine, pH 5.5, at 37 degrees C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C. acidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.

Studies on the secondary structure of 5.8 S rRNA from a thermophile, Thermomyces lanuginosus.

The nucleotide sequence of ribosomal 5.8 S RNA from a thermophilic fungus, Thermomyces lanuginosus, was determined to be (formula: see text). The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques. The results generally supported the "burp gun" model previously proposed for all 5.8 S rRNAs (Nazar, R. N., Sitz, T. O., and Busch, H. (1975) J. Biol. Chem. 250, 8591-8597) and were inconsistent with a recently suggested "cloverleaf" configuration (Luoma, G. A., and Marshall, A. G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4901-4905). Theoretical considerations of the overall stability also appear to favor the former estimate. As previously observed with other 5.8 S RNAs the sequence of T. lanuginosus RNA is strikingly homologous with other species; it differs in only 13 positions from that of yeast. When compared to yeast, the differences appear not to contribute to the stability of the secondary structure but probably lead to a more stable 5.8 S-25 S rRNA interaction in the large ribosomal subunit. A general comparison of T. lanuginosus 5.8 S RNA with all known 5.8 S RNA sequences indicates that, although modified nucleotides differ significantly between species, they are always located in four highly conserved regions of the 5.8 S molecule, probably contributing to the unique character of these very essential sequences.

Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus.

Primate cells harboring the Epstein-Barr virus (EBV) genome synthesize large amounts of two small RNAs:EBER 1 and EBER 2 (EBV-encoded RNA). These RNAs are approximately 180 nucleotides long, possess 5' pppA termini, and lack poly(A). They have different T1 and pancreatic RNase digestion fingerprints. They are not found in normal B lymphocytes, in transformed B lymphocytes that lack EBV DNA, in T lymphocytes transformed by Herpesvirus ateles, or in a variety of other nonlymphoid mammalian cells. Hybridization analyses indicate that EBER 1 and EBER 2 are encoded by the EcoRI-J fragment of EBV (B95-8) DNA. In vivo both RNAs are associated with protein(s), allowing their specific precipitation by the systemic lupus erythematosus-associated antibody anti-La. The La antigen in uninfected mammalian cells consists of a heterogeneous class of small ribonucleoprotein particles, some of whose RNA components exhibit sequence homology with a highly repetitive, interspersed class of human DNA designated the Alu family. Possible functions for EBER 1 and EBER 2 in infection and cell transformation by EBV and their potential relationship to the pathogenesis of systemic lupus erythematosus are discussed.

Nucleotide sequence of wheat chloroplastid 4.5 S ribonucleic acid. Sequence homologies in 4.5 S RNA species.

The nucleotide sequence of wheat (Triticum aestivum L.) chloroplastid ribosome-associated 4.5 S RNA is U-A-A-G-G-U-G-A-G-C-G-G-C-G-A-G-A-C-G-A-G-C-C-G-U-U-U-A-A-A-U-A-G-G-U-G-U-C-A-A -G-U-G-G-A-A-G-U-G-C-A-G-U-G-A-U-G-U-A-U-G-C-A-G-C-U-G-A-G-G-C-A-U-C-C-U-A-A-C- G-A-A-C-G-A-A-C-G-A-U-U-U-G-A-A-COH. The sequence is highly conserved among chloroplastid 4.5 S RNAs but not related to either the 5 S or 5.8 S rRNA species. When compared with 4.5 S RNAs from other origins, the results indicate that this molecule bears little sequence homology to the mammalian nuclear 4.5 S RNA but suggest that chloroplastid 4.5 S RNA may be related to the bacerial 4.5 S species. An estimate for the secondary structure of the wheat 4.5 S molecule was deduced from products of limited pancreatic and T1 ribonuclease digestion, and similarities with the Escherichia coli 4.5 S RNA are discussed.

Isolation and sequence determination of 5'-terminal oligonucleotide fragments of RNA transcripts synthesized by bacteriophage T3-induced RNA polymerase from T3 DNA.

The nucleotide sequence of the 5'-terminal oligonucleotides produced by pancreatic RNase digestion of bacteriophage T3 RNA polymerase (EC 2.7.7.6) transcripts of T3 DNA has been determined. The sequence determination is based upon a simple isolation procedure for the 5'-terminal oligonucleotides. This procedure involves treatment of pancreatic RNase digests of alpha 32P-labeled T3 RNA polymerase transcripts with bovine brain exoribonuclease to remove oligonucleotides with free 5'-hydroxyl termini and then chromatographing the products on hydroxylapatite to resolve the remaining oligonucleotides having 5'-phosphate termini. By application of standard two-dimensional separation and sequence techniques, the major 5'-end sequences deduced were pppGpGpGpApGpApGpApY(Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp. In addition, the sequences of other minor 5'-terminal oligonucleotides observed on homochromatograms were also determined. The sequences of these 5'-oligonucleotides were pppGpGpGpApApCpY, pppGpGpGpApApUpY, pppGpGp(2-4 Gp, 2-3 ApGp)..., and pppGpGpGp.... These results demonstrate that T3 phage-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains.

Nucleotide sequence of the simian virus 40 HindII + III restriction fragment I (fourth part of the T antigen gene).

The HindII + III restriction fragment I (Hind-I) from simian virus 40 DNA represents 4.96% of the genome and maps in the early transcription region. Hind-I is an internal segment of the A gene and its information is expressed as part of the early 19-S mRNA, which codes for T antigen. We report here the nucleotide sequence of the 259-base-pair Hind-I fragment. The sequence was determined and confirmed by RNA and DNA sequencing methods: by analysis of oligonucleotides resulting from T1 and pancreatic RNase digestion of labeled RNA transcribed from SV40 DNA with Escherichia coli DNA-dependent RNA polymerase, by partial degradation of RNA transcripts with snake venom phosphodiesterase, and by base-specific chemical degradation of 5'-end-labeled subfragments of Hind-I according to the procedure of Maxam and Gilbert. Multiple triplets corresponding to termination codons occur in two of the three reading frames of the DNA strand that has the same polarity as early mRNA. The open reading frame connects in phase with the one of the Hind fragments flanking Hind-I, and the amino acid sequence specified by Hind-I lies in the middle part of the large-T antigen. Some features of the primary nucleotide sequence and of early transcription are discussed.

Construction and characterization of a plasmid containing a nearly full-size DNA copy of bacteriophage MS2 RNA

An apparently full-length complementary DNA copy of in vitro polyadenylated MS2 RNA was synthesized with avian myeloblastosis virus RNA-dependent DNA polymerase. After the MS2 RNA template was removed from the complementary DNA strand with T 1 and pancreatic RNase digestion, the complementary DNA became a good template for the synthesis of double-stranded MS2 DNA with Escherichia coli DNA polymerase I. We then constructed molecular chimeras by inserting the double-stranded MS2 DNA into the PstI restriction endonuclease cleavage site of the E. coli plasmid pBR322 by means of the poly(dA)· poly(dT) tailing procedure. An E. coli transformant carrying a plasmid with a nearly full-length MS2 DNA insertion, called pMS2-7, was chosen for further study. Correlation between the restriction cleavage site map of pMS2-7 DNA and the cleavage map predicted from the primary structure of MS2 RNA, and nucleotide sequence analysis of the 5′ and 3′ end regions of the MS2 DNA insertion, showed that the entire MS2 RNA had been faithfully copied, and that, except for 14 nucleotides corresponding to the 5′-terminal sequence of MS2 RNA, the fulllength DNA copy of the viral genetic information had been inserted into the plasmid. Restriction endonuclease analysis of the chimera plasmid DNA also revealed the presence of an extra DNA insertion which was identified as the translocatable element IS1 ‡ ‡ Abbreviations used: IS, insertion sequence; dsDNA, double-stranded DNA; cDNA, complementary DNA; AMV, avian myeloblastosis virus. (see following paper).

Interaction of a limited set of proteins with different mRNAs and protection of 5'-caps against pyrophosphatase digestion in initiation complexes.

A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.

Nucleotide Sequence of the Simian Virus 40 Hind II + III Restriction Fragment J and the Total Amino Acid Sequence of the Major Structural Protein VP1

The HindII + III restriction fragment J (Hind-J) represents 4.58% of the simian virus 40 genome. The information present in Hind-J is expressed as part of the major, late 16-S messenger RNA, which codes for the structural protein VP1. The nucleotide sequence of the 240-base-pairs-long Hind fragment J has been determined by analysis of each oligonucleotide from both strands resulting from T1 or pancreatic RNase digestion of RNA transcribed from the DNA and from RNase digestion of ribo-substituted DNA. Large oligonucleotide blocks which could be constructed mainly on the basis of complementarity were subsequently ordered by partial chemical degradation of terminally labeled DNA. This direct DNA sequencing approach also completely confirmed the results obtained by both aforementioned RNase degradation methods. In the strand with the same polarity as the late mRNA, triplets corresponding to termination codons are present in two of the three reading frames. The one open reading frame connects in phase with the open reading frame of the neighboring HindII/ III fragments K, F and G, which have been published previously and which together with Hind-J span the total VP1 gene. Some features of the primary nucleotide sequence of this VP1 gene and the derived VP1 amino acid sequence are discussed.

Characterization of the 3′ Termini of the RNAs of Cowpea Mosaic Virus

A sequence of polyadenylic acid, homogeneous in composition but heterogeneous in length, was isolated from complete pancreatic RNase digests of both middle and bottom RNAs of cowpea mosaic virus. The polyadenylic acid was 3'-terminal and occurred once per molecule. A fragment consisting of the polyadenylic acid and approximately the next 25 nucleotides could be isolated from complete T1 RNase digests of either RNA. The region adjacent to the polyadenylic acid in both RNAs was rich in pyrimidines. The mobilities of the fragments in 12.4% polyacrylamide-8 M urea gels were used to estimate their lengths and to calculate number average and weight average molecular weights.

Two initiation sites for adenovirus 5.5S RNA.

Adenovirus specific 5.5S RNA is heterogeneous at its 5' terminus. Complete pancreatic RNase digests of the RNA reveal a 5' terminal oligonucleotide (pp)pApGpCp in addition to the major 5' terminal (pp)pGpGpGpCp (1). Both 5' termini are detected early as well as late after adenovirus infection. In isolated nuclei, alpha-amanitin inhibits all 5.5S RNA transcription at a concentration of 200 microgram/ml, indicating that both initiation sites are recognized by RNA polymerase III.

RNase T1-resistant oligonucleotides of Akv-1 and Akv-2 type C viruses of AKR mice

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides derived from the genomes of Akv-1 and Akv-2 type C viruses of AKR mice. The fingerprints of these two viruses look identical. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses were indistinguishable. These observations are consistent with, but not proof of, the possible identity of the genomes of the Akv-1 and Akv-2 viruses and, thus, of the viral genetic material believed to comprise the Akv-1 and Akv-2 loci of AKR mice.

Interaction of rabbit reticulocyte ribosomes with bacteriophage f1 mRNA and of Escherichia coli ribosomes with rabbit globin mRNA

We have compared the behavior of a prokaryotic mRNA in a eukaryotic ribosome binding system and of a eukaryotic mRNA in a prokaryotic ribosome binding system. Using (32)P- and (125)I-labeled bacteriophage f1 mRNA, we have shown that rabbit reticulocyte 80S ribosomes can protect specific sequences from pancreatic RNase digestion, including those sequences protected by Escherichia coli ribosomes. We have also found that E. coli ribosomes fail to protect any region of (125)I-labeled globin mRNA. Iodination of the mRNA appeared to have little or no effect on the specificity of binding or protection by the ribosomes of either system.The eukaryotic and prokaryotic systems differ markedly in the ability of the small ribosomal subunits to protect mRNA from nuclease digestion. The regions of phage f1 mRNA protected by E. coli 30S subunits are virtually identical to those protected by the 70S ribosomes. By contrast, rabbit reticulocyte 40S subunits protect substantially larger fragments of mRNA from nuclease digestion than do the 80S ribosomes. These 40S-protected fragments are specific in the case of globin mRNA and overlap the shorter region protected by the 80S ribosomes. However, the 40S-protected fragments of phage f1 mRNA were found to be extremely heterogeneous, reflecting perhaps an important difference between the initial interactions made by these two mRNAs with the ribosomes.

In vitro synthesis of a high molecular weight virion-associated RNA by vaccinia.

Although the bulk of RNA synthesized in vitro by vaccinia virus is 8 to 12 S, a small amount of high molecular weight RNA can be detected. This RNA is virion-associated and is not extruded from the virus as high molecular weight RNA. It is sensitive to pancreatic RNase digestion in high salt, has a density in neutral CS2SO4 of 1.68 g ml-1 and remains large after digestion with DNase or denaturation in dimethyl sulfoxide. In the presence of high concentrations of virus in the in vitro RNA polymerase reaction, pulse-labeling experiments indicate an RNA sedimenting heterogeneously between 20 and 30 S. Pulse-chase experiments indicate that a fraction of this high molecular weight RNA can be chased into RNA sedimenting at 8 to 12 S. Cleavage into smaller fragments is not dependent on continued RNA synthesis but does require ribonucleoside triphosphates. In the presence of ethidium bromide, the RNA is not cleaved.