RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

Two concomitant base substitutions in the putative replicase genes of tobacco mosaic virus confer the ability to overcome the effects of a tomato resistance gene, Tm-1.

Abstract: A resistance-breaking strain of tobacco mosaic virus (TMV), Ltal, is able to multiply in tomatoes with the Tm-1 gene, unlike its parent strain, L. Comparison of the genomic sequences of L and Lta1 revealed two base substitutions resulting in amino acid changes in the 130 and 180 kd proteins: Gln-979 --> Glu and His-984 --> Tyr. To clarify their involvement in the resistance-breaking property of Lta1, the two substitions were introduced into L by an in vitro transcription system to generate a mutant strain, T1. T1 multiplied in Tm-1/Tm-1 tomatoes with symptoms as did Lta1. Two additional mutant strains were constructed, each of which had one base substitution which caused a His-984 --> Tyr change (T2) or a Gln-979 --> Glu change (T3). T3 multiplied in tomato plants and protoplasts with the Tm-1 gene, indicating that the single base substitution is sufficient to overcome the resistance. T2 also multiplied, but its multiplication was greatly decreased. Although no sequence changes were detected in any progeny viruses recovered from plants without the Tm-1 gene, progeny viruses recovered from T2- or T3- inoculated Tm-1/Tm-1 tomatoes contained in most cases viruses with additional second base substitutions. They caused amino acid changes near the mutagenized residues, suggesting that the ability of T3 to overcome the resistance is not the same as that of Lta1. Sequencing of the genomic RNAs of other independently isolated resistance-breaking strains revealed the same two base substitutions found in the Lta1 RNA. These observations suggest that the two concomitant base substitutions, and possibly also the resulting amino acid changes, guarantee successful replication of these TMV strains in tomatoes containing the Tm-1 gene. A strong correlation was found between the ability to overcome the resistance and a decrease in local net charge, suggesting the involvement of an electrostatic interaction between the viral 130 and 180 kd proteins and a putative host resistance factor.

Rolling-Circle RNA Synthesis: Circular Oligonucleotides as Efficient Substrates for T7 RNA Polymerase.

Abstract: The synthesis of ribonucleic acid strands by RNA polymerase enzymes has long been known to initiate specifically at well-defined promoter sequences.1 For example, the T7 RNA polymerase enzyme initiates RNA strand synthesis with a pppG residue immediately downstream from a conserved ~17-base-pair duplex DNA sequence.2 This fact has been used in the “runoff transcription” method for laboratory synthesis of sequence-defined oligoribonucleotides, in which DNA strands which contain the double-stranded promoter at one end serve as templates for RNA synthesis.3

Poliovirus Requires a Precise 5′ End for Efficient Positive-Strand RNA Synthesis

Abstract: Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5' end, which are deleted during replication to generate authentic viral genomes. We removed those 5'-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5' ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5' end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.