Localization, mobility and fidelity of retrotransposed Group II introns in rRNA genes.

We previously showed that the group II Lactococcus lactis Ll.LtrB intron could retrotranspose into ectopic locations on the genome of its native host. Two integration events, which had been mapped to unique sequences, were localized in the present study to separate copies of the six L.lactis 23S rRNA genes, within operon B or D. Although further movement within the bacterial chromosome was undetectable, the retrotransposed introns were able to re-integrate into their original homing site provided on a plasmid. This finding indicates not only that retrotransposed group II introns retain mobility properties, but also that movement occurs back into sequence that is heterologous to the sequence of the chromosomal location. Sequence analysis of the retrotransposed introns and the secondary mobility events back to the homing site showed that the introns retain sequence integrity. These results are illuminating, since the reverse transcriptase (RT) of the intron-encoded protein, LtrA, has no known proofreading function, yet the mobility events have a low error rate. Enzymatic digests were used to monitor sequence changes from the wild-type intron. The results indicate that retromobility events have approximately 10(-5) misincorporations per nucleotide inserted. In contrast to the high RT error rates for retroviruses that must escape host defenses, the infrequent mutations of group II introns would ensure intron spread through retention of sequences essential for mobility.

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.

Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Multiple metal ions drive DNA association by PvuII endonuclease.

Restriction enzymes serve as important model systems for understanding the role of metal ions in phosphodiester hydrolysis. To this end, a number of laboratories have reported dramatic differences between the metal ion-dependent and metal ion-independent DNA binding behaviors of these systems. In an effort to illuminate the underlying mechanistic details which give rise to these differences, we have quantitatively dissected these equilibrium behaviors into component association and dissociation rates for the representative PvuII endonuclease and use these data to assess the stoichiometry of metal ion involvement in the binding process. The dependence of PvuII cognate DNA on Ca(II) concentration binding appears to be cooperative, exhibiting half-saturation at 0.6 mM metal ion and yielding an n(H) of 3.5 +/- 0.2 per enzyme homodimer. Using both nitrocellulose filter binding and fluorescence assays, we observe that the cognate DNA dissociation rate (k(-)(1) or k(off)) is very slow (10(-)(3) s(-)(1)) and exhibits a shallow dependence on metal ion concentration. DNA trap cleavage experiments with Mg(II) confirm the general irreversibility of DNA binding relative to cleavage, even at low metal ion concentrations. More dramatically, the association rate (k(1) or k(on)) also appears to be cooperative, increasing more than 100-fold between 0.2 and 10 mM Ca(II), with an optimum value of 2.7 x 10(7) M(-)(1) s (-)(1). Hill analysis of the metal ion dependence of k(on) indicates an n(H) of 3.6 +/- 0.2 per enzyme dimer. This value is consistent with the involvement in DNA association of two metal ions per subunit active site, a result which lends new strength to arguments for two-metal ion mechanisms in restriction enzymes.

Dissecting the metal ion dependence of DNA binding by PvuII endonuclease.

Divalent cations can provide an effective means of modulating the behavior of nucleic acid binding proteins. As a result, there is strong interest in understanding the role of metal ions in the function of both nucleic acid binding proteins and their enzymes. We have applied complementary fluorescence spectroscopic and nitrocellulose filter binding assays to quantitate the role of metal ions in mediating DNA binding and sequence specificity by the representative PvuII endonuclease. At pH 7.5 in the presence of the catalytically nonsupportive Ca(II), this enzyme binds the PvuII target sequence with a K(d) of 50 pM. Under strict metal-free conditions, the enzyme exhibits a K(d) of only 300 nM for the cognate sequence, an affinity which is weak relative to those measured for other systems in the absence of metal ions. This represents a 6000-fold increase in PvuII affinity for cognate DNA upon the addition of Ca(II). The pH dependences of both metal ion-dependent and metal ion-independent DNA binding are remarkably shallow throughout the physiological range; other characterized restriction enzymes exhibit more pronounced pH dependences of DNA binding even in the absence of metal ions. Similar measurements with noncognate sequences indicate that divalent metal ions are not important to nonspecific DNA binding; K(d) values are approximately equal to 200 nM throughout the physiological pH range, a behavior shared with other endonucleases. While some of these results extend somewhat the range of expected behavior for restriction enzymes, these results indicate that PvuII endonuclease shares with other characterized systems a mechanism by which cognate affinity and sequence discrimination are most effectively achieved in the presence of divalent metal ions.