RNA splicing in the T-even bacteriophage

Group 1 introns, first demonstrated in the nuclear large rRNA of Tetrahymena thermophila and subsequently in many yeast, fungal mitochondrial, and chloroplast precursor RNAs, are capable of intron excision and exon ligation in vitro, although this process occurs much more rapidly in vivo. The discovery and characterization of a similar intron in the T4 phage thymidylate synthase gene (td) led to the finding of additional group 1 introns in other T4 genes and in genes of the related T2 and T6 phages. Because protein factors are not required in the splicing of group 1 introns in vitro, it has been postulated that the precursor RNA can assume a critical conformation enabling it to undergo site-specific autocatalytic cleavage and ligation (self-splicing). By means of site-directed mutation, it has been shown unequivocally that several sequence elements in the Tetrahymena rRNA intron are involved in the formation of base-paired stem structures that are essential for the self-splicing process. These sequence elements have been demonstrated in other eukaryotic group 1 introns, as well as in the td intron. In this brief review we shall describe the biochemical and structural properties of the td intron in relation to other newly found phage introns. The interesting implications arising from these revelations will also be discussed.     

Sedimentation coefficient of T-even bacteriophage DNA

The sedimentation coefficient of T2 phage DNA has been studied by zone centrifugation in sector-shaped preparative centrifuge tubes over a concentration range of 0.02–2.0 μg./ml. DNA. These results have been compared to a similar study in the analytical centrifuge of T4 DNA over the range of 0.50–5.75 μg./ml. DNA. A value for the sedimentation coefficient of 60.7 ± 1.8 S. was obtained by the first method and a value of 61.3 ± 1.5 S. by the second.     

Apparent non-involvement of transfer RNA nucleotidyltransferase in the biosynthesis of Escherichia coli suppressor transfer RNAs

Transfer RNA nucleotidyltransferase has previously been shown to be required for the normal growth of Escherichia coli and for the biosynthesis of some bacteriophage T4 tRNAs. In order to obtain information about the involvement of this enzyme in E. coli tRNA biosynthesis we have measured the level of activity of suppressors 1 to 6 in strains carrying either a cca⁺ or cca allele. We found that cca strains, deficient in tRNA nucleotidyltransferase, contained the same amount of suppressor activities as the wild-type cca⁺ strains as determined by suppression of nonsense mutations in both E. coli alkaline phosphatase and in genes of bacteriophage T4. The results suggest that tRNA nucleotidyltransferase is not required for the biosynthesis of tRNAs specified by suppressors 1 to 6.     

Evolution of methionine initiator and phenylalanine transfer RNAs

Summary Sequence data from methionine initiator and phenylalanine transfer RNAs were used to construct phylogenetic trees by the maximum parsimony method. Although eukaryotes, prokaryotes and chloroplasts appear related to a common ancestor, no firm conclusion can be drawn at this time about mitochondrial-coded transfer RNAs. tRNA evolution is not appropriately described by random hit models, since the various regions of the molecule differ sharply in their mutational fixation rates. Hot mutational spots are identified in the TC, the amino acceptor and the upper anticodon stems; the D arm and the loop areas on the other hand are highly conserved. Crucial tertiary interactions are thus essentially preserved while most of the double helical domain undergoes base pair interchange. Transitions are about half as costly as transversions, suggesting that base pair interchanges proceed mostly through G-U and A -C intermediates. There is a preponderance of replacements starting from G and C but this bias appears to follow the high G + C content of the easily mutated base paired regions.     

Structural elements of the 3'-terminal coat protein binding site in alfalfa mosaic virus RNAs.

The 3'-terminal of the three genomic RNAs of alfalfa mosaic virus (AIMV) and ilarviruses contain a number of AUGC-motifs separated by hairpin structures. Binding of coat protein (CP) to such elements in the RNAs is required to initiate infection of these viruses. Determinants for CP binding in the 3'-terminal 39 nucleotides (nt) of AIMV RNA 3 were analyzed by band-shift assays. From the 5'- to 3'-end this 39 nt sequence contains AUGC-motif 3, stem-loop structure 2 (STLP2), AUGC-motif 2, stem-loop structure 1 (STLP1) and AUGC-motif 1. A mutational analysis showed that all three AUGC-motifs were involved in CP binding. Mutation of the A- and U-residues of motifs 1 or 3 had no effect on CP binding but similar mutations in motif 2 abolished CP binding. A mutational analysis of the stem of STLP1 and STLP2 confirmed the importance of these hairpins for CP binding. Randomization of the sequence of the stems and loops of STLP1 and STLP2 had no effect on CP binding as long as the secondary structure was maintained. This indicates that the two hairpins are not involved in sequence-specific interactions with CP. They may function in a secondary structure-specific interaction with CP and/or in the assembly of the AUGC-motifs in a configuration required for CP binding.     

Rotational effects in quasielastic laser light scattering from T-even bacteriophage.

We have applied a theory of dynamic light scattering from large anisotropic particles, developed by Aragón and Pecora [J. Chem. Phys. 66 , 2506–2516 (1977)] to calculate the scattering expected from T-even phage models. The results indicate that the off-center rotation of the massive virus head with respect to the center of frictional resistance introduces significant rotational contributions to the light-scattering time autocorrelation function. The effect is particularly important when the fibers of the phage are extended. Reanalysis of previously published data [J. B. Welch III and V. A. Bloomfield, Biopolymers 17 , 2001–2014 (1978)], taking into account rotational corrections, confirms the equality of molecular weights of the slow- and fast-sedimenting forms of T2L bacteriophage.     

A role for ribonuclease III in synthesis of bacteriophage T4 transfer RNAs.

Synthesis of T4 tRNA^Gln depends on normal levels of $\text{Escherichia}\text{coli}$ ribonuclease III. Infection of cell strains carrying a mutation in the gene for this enzyme resulted in severe depression in tRNA^Gln production, as revealed by chemical and suppressor tRNA analyses. The remaining seven T4 tRNAs were synthesized in the mutant cells. The requirement of ribonuclease III for synthesis of tRNA^Gln points to an essential cleavage by the enzyme of a precursor RNA containing tRNA^Gln.     

Structural aberrations in T-even bacteriophage. IX. Effect of mixed infection on the production of giant bacteriophage.

To date, the production of T-even bacteriophage with giant heads has been achieved in two ways: (i) by use of canavanine-arginine treatment of Escherichia coli B cultures infected by wild-type bacteriophage (Cummings and Bolin, Bacteriol. Rev. 40:314-359, 1976; Cummings et al., Virology 54:245-261, 1973), which give a size distribution of giants that is phage specific (Cummings et al., Virology 54:245-261, 1973); and (ii) by infection with certain missense mutants of T4D gene 23 (Doermann et al., J. Virol. 12:374-385, 1973; ICN-UCLA Symposium on Molecular Biology, p. 243-285, 1973) or temperature-sensitive mutants of gene 24 (Aebi et al., J. Supramol. Struct. 2:253-275, 1974; Biljenga et al., J. Mol. Biol. 103:469-498, 1976). We now report the effect of mixed infection with several mutants of T4D on both the production and the size of giant bacteriophage. We found that gene 24 mutant is a critical partner for the production of giants. Infection using T4.24 mutants together with either T4.23 mutants, T4B+ or T6+ led to the formation of giants with heads 10- to 14-fold longer than normal-length heads. Infection with amber 24-bypass 24 double mutants of T4D led to the production of giants when gene 23 mutant was used to co-infect. Addition of canavanine to the co-infected cultures could alter the size distribution of giants, depending on which phage were used to coinfect. Gene 22 mutants had a modifying effect on these results. In the absence of canavanine co-infection with gene 22 mutants prevented the production of giants, and in the presence of canavanine giants of 1.5 to 5 head lengths were found. We have interpreted these results to mean that critical concentrations of gene products 22, 23, and 24 interact to control head length in T-even bacteriophage.     

Mutant prohead RNAs in the in vitro packaging of bacteriophage phi 29 DNA-gp3.

The 174-base prohead RNA encoded by bacteriophage phi 29 of Bacillus subtilis, essential for packaging of the DNA-gp3 (DNA-gene product 3) complex, was expressed efficiently from the cloned gene. Computer programs for RNA structure analysis were used to fold hypothetical RNA mutants and thus to target mutagenesis of the RNA for studies of structure and function. Five mutants of the RNA were then produced by oligonucleotide-directed mutagenesis that were altered in the primary sequence at selected sites; two of these mutants were predicted to be altered in secondary structure from a model established previously by a phylogenetic analysis. The binding of the 32P end-labeled mutant RNAs to RNA-free proheads was comparable with that of the wild-type RNA. However, the capability of the mutant RNAs to reconstitute RNA-free proheads for DNA-gp3 packaging in the defined in vitro system and for assembly of phage in RNA-free extracts was variable, depending upon the alteration. Changes of highly conserved bases that retained the predicted secondary structure of the RNA model were tolerated to a much greater extent than changes predicted to alter the RNA secondary structure.     

On the biosynthesis of 5-methoxyuridine and uridine-5-oxyacetic acid in specific procaryotic transfer RNAs

The uridine-5-0-derivatives, 5-methoxyuridine (mo5U) and uridine-5-oxyacetic acid (cmo5U) occupy the first position of anticondons in certain tRNA species of B. subtilis and E. coli, respectively. Here we present experimental evidence showing that both modifications are derived from a common precursor, 5-hydroxyuridine. Incompletely modified tRNASer and tRNAVal from E. coli met- rel-. All five tRNAs accepted methyl groups from S-adenosylmethionine with B. subtilis extracts in vitro and mo5U was formed. In B. subtilis tRNAs the mo5U was proved to be at the specific site; in E. coli tRNAVal the mo5U was demonstrated to be present in the oligonucleotide that comprises the anticodon. In submethylated E. coli tRNAVal,5-hydroxyuridine was detected whereas considerable amounts of cmo5U were lacking.