nrdB Intron Research Articles

Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII

Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

Homing endonuclease I-TevIII: dimerization as a means to a double-strand break

Homing endonucleases are unusual enzymes, capable of recognizing lengthy DNA sequences and cleaving site-specifically within genomes. Many homing endonucleases are encoded within group I introns, and such enzymes promote the mobility reactions of these introns. Phage T4 has three group I introns, within the td, nrdB and nrdD genes. The td and nrdD introns are mobile, whereas the nrdB intron is not. Phage RB3 is a close relative of T4 and has a lengthier nrdB intron. Here, we describe I-TevIII, the H–N–H endonuclease encoded by the RB3 nrdB intron. In contrast to previous reports, we demonstrate that this intron is mobile, and that this mobility is dependent on I-TevIII, which generates 2-nt 3′ extensions. The enzyme has a distinct catalytic domain, which contains the H–N–H motif, and DNA-binding domain, which contains two zinc fingers required for interaction with the DNA substrate. Most importantly, I-TevIII, unlike the H–N–H endonucleases described so far, makes a double-strand break on the DNA homing site by acting as a dimer. Through deletion analysis, the dimerization interface was mapped to the DNA-binding domain. The unusual propensity of I-TevIII to dimerize to achieve cleavage of both DNA strands underscores the versatility of the H–N–H enzyme family.

Restoration of mRNA Splicing by a Second-Site Intragenic Suppressor in the T4 Ribonucleotide Reductase (Small Subunit) Self-Splicing Intron

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. Thirty-one mutants causing splicing defects in the nrdB intron were isolated. Twenty-three EMS-induced revertants for these 31 primary mutants were isolated by the strategic usage of the white halo plaque phenotype. We mapped these revertants by marker rescue using subclones of the nrdB gene. Some of these second-site mutations mapped to regions currently predicted by the secondary structure model of the nrdB intron. One of these suppressor mutants (nrdB753R) was found to be intragenic by marker rescue with the whole nrdB gene. However, this mutation failed to map within the nrdB intron. Splicing assays showed that this pseudorevertant restored splicing proficiency of the nrdB primary mutation to almost wild-type conditions. This is the first example of a mutation within the exons of a gene containing a self-splicing intron that is capable of restoring a self-splicing defect caused by a primary mutation within the intron. In addition, two other suppressor mutations are of interest (nrdB429R and nrdB399R). These suppressors were able to restore their primary 5′ defect but in turn create a 3′ splicing defect. Both of these revertants mapped in different regions of the intron with respect to their primary mutations.

Isolation and Characterization of EMS Induced Splicing Defective Point Mutations within the Intron of thenrdB Gene of Bacteriophage T4

ThenrdBgene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base-pair self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. The screening, isolation, and mapping of 31nrdB intron mutations were conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. These intron mutations cluster towards the ends, mainly the 3′ end, and show a defect in self-splicing. These mutations map in regions of conserved structural elements, thus supporting secondary structure predictions. A distinct pattern of clustering is observed with the highest number of mutations mapping within three of the smaller regions (A, C, and D) of the nrdB intron and no mutations mapping in the largest (B) region. The highest density of mutations mapped in the smallest region (C) of the intron, containing only 96 bases, thus showing a distinct pattern of clustering within the catalytic core.

Functional and sequence analysis of splicing defective nrdB mutants of bacteriophage T4 reveal new bases and a new sub-domain required for group I intron self-splicing

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 nuclelotide group 1 self splicing intron. In order to study the functional domains for self-splicing of this intron, 23 nrdB splicing defective intron mutants were analysed for both sequence and functional changes. These mutants cluster towards the ends in regions of conserved structural elements of the intron. These 23 mutants have single base changes at 14 different sites. Interestingly two of these sites that seemed to map within the intron are actually located on the flanking exon sequences on both sides of the intron. A high frequency (4/12) of the mutation sites are in bases not thought to be base-paired in the standard model of group I intron structure. The mutation sites in pairing regions P3, P7, P8, P9 and between P6[3′] and P7[5′] are identical to changes found in the well studied td (encoding dTMP synthase) intron. However, five new mutation sites (S61, SL1, S29, SL11, SL196 and SL126) are unique to the nrdB intron and disrupt self-splicing. A mutation (S61) in the P7.1 pairing region is especially significant because no mutations have been found in this pairing, thus defining a new sub-domain essential for RNA splicing. Like the td intron, the mutation site in P9 of the nrdB intron is a hot spot for mutations, but unlike td, the nrdB intron does not show a mutational hot spot in the P6[5′] region. Our molecular dissection of the nrdB intron also supports the P9.0 and P10 pairings that have been postulated to help form a complex tertiary structure required to give the RNA sequence its catalytic activity: particularly 3′ splice site selection, cleavage and exon ligation.

A Novel Approach for Isolation and Mapping of Second-Site Revertants of Intron Mutations in a Ribontjcleotide Reductase Encoding Gene (nrdB) of Bacteriophage T4 Using the White Halo Plaque Phenotype

Abstract The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of T4 and eukaryotes. Previously, the nrdB intron mutations, presumably causing splicing defects of the nrdB transcript, were isolated and mapped in or near the nrdB intron. In this communication, we have isolated 181 hydroxylamine-induced revertants for the above primary nrdB intron mutations by strategic usage of the white halo phenotype. Also, we mapped these revertant mutations by marker rescue with subclones of the nrdB gene. Some of the second site mutations were mapped to regions predicted by the secondary structure model of the nrdB intron. To investigate the involvement of protein factors facilitating splicing of the nrdB transcript, we attempted to isolate extragenic revertants of td-nrdB double mutants by utilizing hydroxylamine mutagenesis. Mapping and sequencing of the suppressor mutations in the extragenic revertants revealed that the second-site mutations are in the frd gene, coding for dihydrofolate reductase. Splicing assays showed that these suppressor mutations do not reverse the splicing defects of td-nrdB mutants, but only affect the halo phenotype, most likely by altering the pyrimidine nucleotide metabolism.

A Novel Approach for Isolation and Mapping of Intron Mutations in a Ribonucleotide Reductase Encoding Gene ( nrdB) of Bacteriophage T4 Using the White Halo Plaque Phenotype

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of 14 and alkaxyotes. The screening, isolation and mapping of the nrbB intron mutations was conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. We have isolated 159 hydroxylamine-induced nrdB mutants, determined which mutations are in nrdB by marker rescue with clones of the nrdB gene and have mapped these mutations by marker rescue using subclones of the nrdB intron. Thirty out of the 159 nrdB mutations are in or near the intron. These mutations cluster towards the ends, mainly the 3′ end. We have performed deletion mapping to further map mutations in the 3′ end of the intron. The mutations map in regions of conserved structural elements, thus supporting secondary structure predictions similar to those of the well studied td intron in the T4 gene coding for thymidylate synthase.

Translation across the 5′‐splice site interferes with autocatalytic splicing

The bacteriophage T4 nrdB gene, encoding the ribonucleotide reductase small subunit, contains a self-splicing group IA2 intron with an ochre codon in frame with the preceding exon sequence. The stop codon was changed to an amino acid codon and splicing efficiency was compared with that of the wild type in the presence and absence of translation. In vivo the mutant has a much lower efficiency for producing a mature transcript than the wild type. Also, the relative production of the full-length translation product is correspondingly lower in the mutant than in the wild type. These results confirm the importance of the stop codon, which spans the splice site of the nrdB intron. The occurrence of stop codons in 56 group I introns in protein-encoding genes was investigated. In 33 of those translation is terminated upstream of the first common elements of the catalytic core, of group I introns. In the rest translation is terminated in intron regions outside the heart of the catalytic core, with one exception. Our observations suggest that in situations where transcription and translation are coupled events there has been an evolutionary pressure to preserve stop codons in the 5'-region of these introns or to prevent translational termination from occurring in vital parts of the introns.

The phage T4 nrdB intron: a deletion mutant of a version found in the wild.

Bacteriophage T4 possesses three self-splicing group I introns. Two of the three introns are mobile elements; the third, in the gene encoding a subunit of the phage nucleotide reductase (nrdB), is not mobile. Because intron mobility offers a reasonable explanation for the paradoxical occurrence of large intervening sequences in a space-efficient eubacterial phage, it is puzzling that the nrdB intron is not mobile like its compatriots. We have discovered a larger nrdB intron in a closely related phage, and we infer from comparative sequence data that the T4 intron is a deletion mutant derived from this larger intron. This larger nrdB intron encodes an open reading frame of 269 codons, which we have cloned and overexpressed. The overexpressed protein shows a dsDNA endonuclease activity specific for the intronless nrdB gene, typical of mobile introns. Thus, we believe that all three introns of T4 are or were mobile "infectious introns" and that they have entered into and been maintained in the phage population by virtue of this efficient mobility.

The inconsistent distribution of introns in the T-even phages indicates recent genetic exchanges.

Group I self-splicing introns are present in the td, nrdB and sunY genes of bacteriophage T4. We previously reported that whereas the td intron is present in T2, T4 and T6, the nrdB intron is present in T4 only. These studies, which argue in favor of introns as mobile genetic elements, have been extended by defining the distribution of all three T4 introns in a more comprehensive collection of T2, T4 and T6 isolates. The three major findings are as follows: First, all three introns are inconsistently distributed throughout the T-even phage family. Second, different T2 isolates have different intron complements, with T2H and T2L having no detectable introns. Third, the intron open reading frames are inherited or lost as a unit with their respective flanking intron core elements. Furthermore, exon sequences flanking sites where introns are inserted in the T4 td, sunY and nrdB genes were determined for all the different T-even isolates studied. Six of eighteen residues surrounding the junction sequences are identical. In contrast, a comprehensive comparison of exon sequences in intron plus and intron minus variants of the sunY gene indicate that sequence changes are concentrated around the site of intron occurrence. This apparent paradox may be resolved by hypothesizing that the recombination events responsible for intron acquisition or loss require a consensus sequence, while these same events result in sequence heterogeneity around the site.

Genes within genes: independent expression of phage T4 intron open reading frames and the genes in which they reside.

The td, nrdB, and sunY introns of bacteriophage T4 each contain a long open reading frame (ORF). These ORFs are preceded by functional T4 late promoters and, in the case of the nrdB intron ORF, a functional middle promoter. Expression of phage-encoded intron ORF-lacZ fusions indicates that these T4 genes are highly regulated. The lack of translation of these ORFs from early pre-mRNAs can be accounted for by the presence of secondary structures that are absent from the late RNAs. Because translation of the intron ORFs could disrupt core structural elements required for pre-mRNA splicing, such regulation may be necessary to allow expression of the genes in which they reside.

Erratum: Variable Occurrence of the nrd B Intron in the T-Even Phages Suggests Intron Mobility

In the report "Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility" by J. Pedersen-Lane and M. Belfort (10 July, p. 182), two sentences were incorrectly printed. On page 182, the sentence beginning at the bottom of the second column should have read, "The td intron is indeed homologous throughout the T-even phages, as demonstrated by hybridization and dideoxy primer-extension analysis of pre-messenger RNA (pre-mRNA) and splice site products (11)." On page 183, in the legend to figure 2, the next-to-last sentence should have read, "Oligonucleotide probes (1 to 4) indicated by arrows are as described (21)."

Infrared Astronomy

In the report " Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility" by J. Pedersen-Lane and M. Belfort (10 July, p.182), two sentences were incorrectly printed. On page 182, the sentence beginning at the bottom of the second column should have read, "The td intron is indeed homologous throughout the T-even phages, as demonstrated by hybridization and dideoxy primer-extension analysis of pre-messenger RNA (pre-mRNA) and splice site products (11)." On page 183, in the legend to figure 2, the next-to-last sentence should have read, "Oligonucleotide probes (1 to 4) indicated by arrows are as described (21)."