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The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

The transfer RNA (tRNA) multigene family comprises 20 amino acid-accepting groups, many of which contain isoacceptors. The addition of isoacceptors to the tRNA repertoire was critical to establishing the genetic code, yet the origin of isoacceptors remains largely unexplored. A model of tRNA evolution, termed "tRNA gene recruitment," was formulated. It proposes that a tRNA gene can be recruited from one isoaccepting group to another by a point mutation that concurrently changes tRNA amino acid identity and messenger RNA coupling capacity. A test of the model showed that an Escherichia coli strain, in which the essential tRNAUGUThr gene was inactivated, was rendered viable when a tRNAArg with a point mutation that changed its anticodon from UCU to UGU (threonine) was expressed. Insertion of threonine at threonine codons by the "recruited" tRNAArg was corroborated by in vitro aminoacylation assays showing that its specificity had been changed from arginine to threonine. Therefore, the recruitment model may account for the evolution of some tRNA genes.

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

Four thousand Escherichia coli clones containing yeast DNA inserted into the plasmid pBR313 have been isolated. Of these, 175 clones were identified as carrying yeast transfer RNA genes. The initial analysis of the inserted transfer RNA genes via the colony hybridization technique with individual radioactive transfer RNA species is reported. The data indicate that yeast transfer RNA genes are not highly clustered, although some clustering exists. In addition, it was observed that the reiteration number of different transfer RNA genes may vary extensively.