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RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5'-leader sequence as well as the 3'-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studied in vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.

We have used genetics as a tool to study the importance of an internal loop (P7) of Escherichia coli RNase P RNA (M1 RNA) in cleavage site selection and the binding of a divalent metal ion(s). The preferred cleavage site on a model tRNA precursor substrate shifted as a result of base-substitutions and deletions within this loop, in particular when changes were introduced at positions directly involved in base-pairing with the 3'-terminal RCCA motif of the substrate. Additionally, these changes in M1 RNA resulted in alterations in the binding of a divalent metal ion(s) in the vicinity of this internal loop as revealed by lead(II)-induced cleavage. From these data we conclude that the structural integrity of the P7 loop is important for both cleavage site selection and divalent metal ion binding. Cross-linking experiments using precursors carrying a 4-thioU immediately 5' of two independent cleavage sites suggest that close contact points between M1 RNA and nucleotides at these cleavage sites depend on the interaction between M1 RNA and the 3'-terminal RCCA motif of the substrate. Our findings further support the view that there are at least two different ways for the tRNA domain of a tRNA precursor to interact with M1 RNA. These results support a model in which base-pairing between M1 RNA and its substrate results in a re-coordination of a divalent metal ion(s) such that cleavage at the correct position is accomplished.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5-5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.

The papova viruses and the human adenoviruses are widely used as a model system to study cell transformation in vitro. In subgroup C human adenoviruses, fragment HpaI-E, which comprises as little as 4.5% of the adenovirus type 5 (ad5) DNA, is sufficient for transformation of rat embryo cells¹. Analysis of messenger RNAs (mRNAs) from the transforming region of adenoviruses type 2 (ad2) has identified several spliced mRN A species²⁻⁴. Promoter mapping studies indicate that the leftmost early region contains two separate transcription units, E1A and E1B (ref. 5) (Fig. 1a). Region E1A is approximately equivalent HpaI-E. The complete nucleotide sequence of the HpaI-E fragment of ad5 was recently reported⁶. However, the spliced nature of early adenovirus mRNAs prevents a prediction of the amino acid sequence of the corresponding polypeptides directly from the DNA sequence. To study the structure of early ad2 mRNAs at the nucleotide level, we have used molecular cloning procedures to amplify the appropriate mRNA sequences. In this report, clones corresponding to the 12S and 13S mRNA from region E1A (Fig. 1c) have been isolated and characterised by hybridisation and sequence analysis. Our results enable us to predict the primary sequence of two related polypeptides from region E1A of human subgroup C adenoviruses.

Antisense RNAs usually are small, highly structured, unstable RNAs that control a varietyof different biological processes. They exert their effect by binding to complementarytarget RNAs and thereby inhibit their functions.

This thesis is concerned with the main features that determine the efficiency of antisense RNAs: their metabolic stability, the binding rate between antisense and target RNA, and the structure of the antisense RNA itself. Three different plasmid-encoded antisense RNAs were studied.

RNA decay in bacteria is complex: a large number of proteins are involved in processing and degradation. My results show that mutations in an Escherichia coli gene, pcnB, encoding a poly(A)polymerase, exert an effect on the replication of antisense RNA-regulated plasmids. PcnB aids in the degradation of the inhibitors of replication, the antisense RNAs. This enzyme can polyadenylate both CopA and RNAI, the antisense RNAs controlling the replication of plasmids R1 and ColE1, respectively. Accelerated pcnB dependent decay of CopA requires processing by an endoribonuclease, RNase E. This cleavage step appears to initiate decay of both CopA and RNA I. In addition, the 3'-5' exoribonucleases PNPase and RNase II can independently degrade RNase E-cleaved CopA.Other ribonucleases are involved in the pcnB dependent degradation since CopA decay is shown (although at lower rates) in the absence of these enzymes.

Analysis of FinP, an antisense RNA controlling plasmid conjugation, indicates astructure consisting of two stem-loops. Stem-loops are preferred motifs in most antisenseRNAs. The binding rate between FinP and its target RNA is in the same range as in otherwell-characterized systems. Upon binding, the target RNA (traJ mRNA) is renderedunstable, probably due to RNase III-dependent cleavages. This destabilization requiresthe presence of the helper protein FinO.