Requires Base Pairing Research Articles

An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation

Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA. By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA. Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop. The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo. These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation.

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.

3′ splice site recognition in S. cerevisiae does not require base pairing with U1 snRNA

The conserved nucleotides 9 and 10 of U1 small nuclear RNA (snRNA) have been proposed to base pair with either 5′ exon or 3′ splice site sequences. In S. pombe, U1 snRNA pairing with the conserved 3′ splice site is required for the first step of splicing and viability. In contrast, we show that S. cerevisiae U1 mutants at positions 9 and 10 are fully functional. Splicing of several genes is normal in these strains, ruling out an essential base pairing between U1 snRNA and 3′ splice sites. U1 snRNA positions 9 and 10 are shown to be involved in 5′ splice site selection through their interaction with exon sequences. Our results demonstrate that some snRNA-pre-mRNA interactions are not evolutionarily conserved and that 3′ splice site recognition occurs by different mechanisms in various organisms.

Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

Linker scanning mutagenesis of the internal ribosome entry site of poliovirus RNA.

The initiation of cap-independent translation of poliovirus mRNA occurs as a result of ribosome entry at an internal site(s) within the 5' noncoding region. A series of linker scanning mutations was constructed to define the genetic determinants of RNA-protein interactions that lead to high-fidelity translation of this unusual viral mRNA. The mutations are located within two distinct stem-loop structures in the 5' noncoding region of poliovirus RNA that constitute a major portion of a putative internal ribosome entry site. On the basis of our data derived from genetic and biochemical assays, the stability of one of the stem-loop structures appears to be essential for translation initiation via internal binding of ribosomes. However, the second stem-loop structure may function in a manner that requires base pairing and proper spacing between specific nucleotide sequences. By employing RNA electrophoretic mobility shift assays, an RNA-protein interaction was detected for this latter stem-loop structure that does not occur in RNAs containing mutations which perturb the predicted hairpin structure. Analysis of in vivo-selected virus revertants, in combination with mobility shift assays, suggests that extensive genetic rearrangement can lead to restoration of 5' noncoding region functions, possibly by the repositioning of specific RNA sequence or structure motifs.

An analysis of sequences stimulating frameshifting in the decoding of gene 10 of bacteriophage T7

The signals necessary for the translational frameshift in the gene 10 message of bacteriophage T7 include the previously identified frameshift site and the 3' non-coding region, over 200 bases downstream. The functional components of the frameshift site are identified in this study and show that the site most probably operates by the retroviral type two site mechanism. However, the base pairing requirements for the first tRNA are much more relaxed after the slip than is seen in other examples. The element at the 3' end of the gene, also necessary for frameshifting, is examined but only the extreme 5' side of the transcriptional terminator stem-loop structure in the 3' non-coding region seems to be required. No simple secondary structural model can explain the involvement of this sequence. The T7 frameshift site can be replaced with either a T3 site or a E. coli dnaX site. Both show higher levels of frameshifting than with the T7 site.

Apurinic sites as common intermediates in mutagenesis.

It is well known that the high fidelity of DNA replication in eucaryotes results in part from the accuracy by which DNA polymerases copy the base sequences in DNA. It is also well known that the reactions of many mutagens and carcinogens with DNA result in the formation of bulky adducts. Experimental evidence indicates that these bulky adducts block DNA synthesis in vitro and in vivo.1 A paradox in mutagenesis is how DNA polymerases, with their stringent requirement for correct base pairing, can accommodate bulky adducts to yield mutations in nascent DNA. One possibility is that the adducts responsible for mutagenesis are labile and form non-bulky intermediary products that are mutagenic. We have proposed that apurinic sites are mutagenic intermediates2 and we have subsequently analyzed their mutagenic potential in vivo and in vitro.

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Capped ribopolymers lacking a sequence complementary to the common 3' end of the influenza virion RNA segments effectively stimulated transcription of these RNAs by the virion-associated transcriptase. Thus, stimulation of transcription results not from hydrogen bonding between the capped RNA and the 3' end of the virion RNA but presumably from a specific interaction of the capped RNA with protein(s) in the transcriptase complex. Although no specific nucleotide sequence was required for priming activity, capped mRNAs with diminished secondary structure were preferred as primers. Inosine-substituted or bisulfite-modified capped reovirus mRNAs were at least 3- to 5-fold more effective as primers than were the native capped mRNAs. On the other hand, inosine substitution or bisulfite treatment of the uncapped form of reovirus mRNAs converted them from essentially inactive species to potent inhibitors of the transcriptase reaction primed by either ApG or globin mRNA. These effects of reduced secondary structure also most probably reflect an interaction of the exogenous RNAs with transcriptase protein(s). The results obtained from screening a series of native uncapped ribopolymers were consistent with inhibitory activity requiring the absence of most hydrogen bonding in the ribopolymer and also suggested that specific structural feature(s) of the nucleotides in the chain were important.

Structural Requirements for Recognition of Escherichia coli Initiator and Non-Initiator Transfer Ribonucleic Acids by Bacterial T Factor

Abstract Escherichia coli formylmethionine tRNA is unable to form a ternary complex with bacterial T factor and GTP, as measured by Sephadex G-50 gel filtration. Treatment of tRNAfMet with sodium bisulfite produces cytidine to uridine base changes in the tRNA structure and greatly enhances its binding affinity for T factor. The ternary complex formed with the bisulfite-modified initiator tRNA has properties analogous to those of EF-Tu·GTP·AA-tRNA complexes formed with non-initiator tRNAs. Enzymatic formylation of modified Met-tRNAfMet completely eliminates its ability to bind to EF-Tu. Unmodified tRNAfMet is unique among the tRNAs sequenced to date in having a non-hydrogen-bonded base at the 5' terminus. Bisulfite-catalyzed conversion of this unpaired cytidine to uridine results in formation of a normal uridine-adenosine base pair at the end of the acceptor stem. In order to determine whether this modification affects T factor recognition, partially modified Met-tRNAfMet was labeled with 32P at the 5' terminus and the fraction capable of forming an EF-Tu·GTP·Met-tRNAfMet complex was separated from unbound Met-tRNAfMet by Sephadex G-100 gel filtration. Terminal nucleotide analysis of the tRNA bound in the complex showed that it was greatly enriched in [5'-32P]uridylate compared to the starting tRNA. Removal of the 5'-phosphate from several normal AA-tRNAs was found to drastically reduce the ability of these tRNAs to form stable ternary complexes with EF-Tu. The requirement for base pairing at the end of the acceptor stem therefore appears to result from a requirement for a specific spatial orientation of the 5'-terminal phosphate for T factor binding. Bisulfite modification of the 3'-terminal CpCpA-OH sequence of Met-tRNAfMet reduces its binding affinity for EF-Tu, indicating that this region of the molecule is also involved in formation of a stable ternary complex. Met-tRNAfMet containing a fully base-paired acceptor stem and normal CpCpA-OH sequence can be completely bound as EF-Tu·GTP·Met-tRNAfMet in the presence of excess T factor, however this ternary complex is less stable than the complexes formed with Met-tRNAmMet, Phe-tRNAPhe, or Gln-tRNAGln, suggesting that other structural features outside of the 3'- and 5'-terminal regions may be involved in T factor recognition of AA-tRNAs.

Sub-cellular localization and characterization of DNA polymerases from rat liver and hepatomas

Summary Previous studies from this laboratory described a correlation between DNA synthesis and DNA polymerase activity in a variety of normal rat tissues, regenerating rat liver and a series of 12 types of rat hepatomas. DNA polymerase in these tissues, isolated by the Mantsavinos procedure, was separated into two peaks of activity by Sephadex G-200 gel filtration. A low molecular weight fraction prefers native DNA primer, is the predominant component of normal rat liver and increases markedly in regenerating liver. A fraction of high molecular weight material which prefers denatured DNA as primer is present to a small extent in normal and regenerating rat liver but increases markedly in fetal rat liver (−6 days from birth), other developing tissues of the rat, rat hepatomas, rat renal tumors and various human cancers. Sub-cellular fractionation of rat liver and selected Morris hepatomas showed that DNA polymerase activity preferring denatured DNA primer is associated with a cytoplasmic smooth membrane fraction in normal liver and is present in the nucleus, as well as with the smooth membranes from hepatoma tissues. The activity with these fractions from the progressed hepatoma 7777 was markedly increased. DNA polymerase preferring native DNA as primer is associated with the nucleus and ribosomes of rat liver and hepatoma. DNA polymerase associated with these sub-cellular fractions from rat liver and hepatoma tissues was partially purified and the properties of the polymerase compared. The ribosomal- and nuclear-associated polymerase of rat liver appears to be the same enzyme in that the physical and enzymatic properties of the purified enzyme are identical. The membrane-associated DNA polymerase, also present in nuclei of hepatomas, has different physical and enzymatic properties than the polymerase associated with both ribosomes and nuclei of liver. After purification both enzymes prefer “activated DNA” as primer. Purified membrane polymerase is free of nuclease activity but purified ribosome polymerase contains a small amount of endonuclease activity that produces single-strand breaks in DNA (“nickase”). All four deoxyribonucleoside triphosphates are required for normal activity of both polymerases but the purified polymerase from ribosomes has about 25% maximal activity in the presence of a single deoxyribonucleoside triphosphate. This activity does not appear to catalyze terminal addition to the primer since experiments with homodeoxyribopolymers demonstrated a requirement for base pairing with the template. We suggest that the physiological function of the polymerase associated with ribosomes and nuclei is in limited polymerization reactions such as DNA repair. The polymerase associated with smooth membranes, and present in the nuclei of proliferating tissues, may function in the replication reaction. Furthermore, we support earlier suggestions ( 18. , 33. ) that the presence of these enzymes in cytoplasm may be due to their origin in this compartment of the cell.