Restore Base Pairing Research Articles

Self-splicing of the group I intron from Anabaena pre-tRNA: requirement for base-pairing of the exons in the anticodon stem.

In the cyanobacterium Anabaena, the precursor to tRNA(Leu) has a 249-nucleotide group I intron inserted between the wobble and second bases of the anticodon; the intron self-splices during transcription in vitro [Xu, M. Q., Kathe, S. D., Goodrich-Blair, H., Nierzwicki-Bauer, S. A., & Shub, D. A. (1990) Science 250, 1566-1570]. By studying splicing of isolated pre-tRNA, we confirm that splicing occurs by the two-step transesterification mechanism characteristic of group I introns, resulting in excision of the intron and accurate ligation of the 5' and 3' exons. The first step, guanosine-dependent cleavage of the phosphodiester bond at the 5' splice site, occurs with kcat congruent to 14 min-1 and kcat/Km = 5 x 10(4) M-1 min-1 (32 degrees C, 15 mM MgCl2), unexpectedly efficient for a small group I intron. (kcat/Km is comparable to that of the Tetrahymena pre-rRNA intron, and kcat is an order of magnitude higher than any previously reported for a group I intron). The second step, ligation of the exons, is so slow (k = 0.3 min-1) that it is rate-limiting for splicing in vitro except at very low guanosine concentrations. Disruption of the base pairs that make up the anticodon stem of the tRNA dramatically reduces the rate of the first step of splicing, while compensatory mutations that restore base pairing generally restore activity. We suggest that the very short P1 helix of this pre-tRNA, with only three base pairs preceding the 5' splice site, is unstable without the additional base pairs in the anticodon stem.(ABSTRACT TRUNCATED AT 250 WORDS)

U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

U2 small nuclear RNA 3' end formation is directed by a critical internal structure distinct from the processing site.

Mature U2 small nuclear RNA is generated by the removal of 11 to 12 nucleotides from the 3' end of the primary transcript. This pre-U2 RNA processing reaction takes place in the cytoplasm. In this study, the sequences and/or structures of pre-U2 RNA that are important for 3' processing have been examined in an in vitro system. The 7-methylguanosine cap, stem-loops I and II, the lariat branch site recognition sequence, the conserved Sm domain, and several other regions throughout the 5' end of U2 RNA have no apparent role in the 3' processing reaction. In fact, deletion of the entire first 104 nucleotides resulted in mini-pre-U2 RNAs which were efficiently processed. Similarly, deletion of the top two-thirds of stem-loop III or mutation of nucleotides in the loop of stem-loop IV had little effect on 3' processing. Most surprisingly, the precursor's 11- to 12-nucleotide 3' extension itself was of relatively little importance, since this sequence could be replaced with completely different sequences with only a minor effect on the 3' processing reaction. In contrast, we have defined a critical structure consisting of the bottom of stem III and the stem of stem-loop IV that is essential for 3' processing of pre-U2 RNA. Compensatory mutations which restore base pairing in this region resulted in normal 3' processing. Thus, although the U2 RNA processing activity recognizes the bottom of stem III and stem IV, the sequence of this critical region is much less important than its structure. These results, together with the surprising observation that the reaction is relatively indifferent to the sequence of the 11- to 12-nucleotide 3' extension itself, point to a 3' processing reaction of pre-U2 RNA that has sequence and structure requirements significantly different from those previously identified for pre-mRNA 3' processing.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression.

Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity

Viroids are single-stranded, covalently closed circular RNA pathogens that can be isolated from certain higher plants afflicted with specific diseases. Their small size (246-375 nucleotides; M(r) 0.8-1.3 x 10(5)) and ability to replicate autonomously make viroids a unique model system in which to study the relationships between the structure of an RNA and its biological function. The demonstrated infectivity of certain cloned viroid cDNAs allows the use of site-specific mutagenesis techniques to probe structure-function relationships suggested by comparative sequence analysis. Several site-specific mutations that disrupt base pairing in either the native structure or secondary hairpin I destroyed the ability of potato spindle tuber viroid cDNA to initiate infection. Alterations in the terminal loops of the native structure also abolished cDNA infectivity. One pseudorevertant, a mutant cDNA containing compensating changes that restore base pairing in the native structure, was marginally infectious; a second pseudorevertant in which base pairing was restored within the stem of secondary hairpin I was not infectious. The behavior of these mutants dramatically demonstrates the effect of remarkably small structural changes on viroid infectivity and emphasizes the importance of the conserved rod-like native structure for viroid function.

Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA.

The U2 snRNP binds to the site of branch formation during splicing of mammalian pre-mRNA in vitro. In Saccharomyces cerevisiae the branch site is within the so-called TACTAAC box (UACUAAC box), an absolutely conserved intron sequence required for splicing. Based on the identification and sequence of a U2 analogue in yeast, a specific base pairing interaction between the UACUAAC box and a highly conserved region of this snRNA can be proposed. To test this hypothesis, we have taken advantage of two mutations constructed previously in the UACUAAC box of an actin-HIS4 fusion. These mutant strains were transformed with stable plasmids bearing U2-like snRNAs into which changes predicted to restore base pairing had been introduced. Allele-specific suppression of biological and biochemical phenotypes was observed in both cases. Recognition of the UACUAAC box thus relies, at least in part, on Watson-Crick base pairing with the yeast U2 analogue.