Full text loading...
Chapter 7 : RNA Processing
Category: Microbial Genetics and Molecular Biology; Environmental Microbiology
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
Most organisms contain between 40 and 50 different transfer RNA (tRNA) molecules that read one or more of the 61 or 62 different sense codons through specific codon-anticodon interactions and position the appropriate amino acid for insertion into the growing polypeptide chain. Additional complexity in the tRNA processing and modification pathway occurs in cases where archaeal tRNA genes contain an intron. The chapter describes introns in archaeal transcripts. The genes for ribosomal RNA (rrn) are located in operons in the archaeal genome and are transcribed to produce multicistronic precursor RNAs. Two early studies used nuclease protection and primer extension assays to define the intermediates generated during processing of the primary rRNA transcript from two canonical rrn operons: the single-rrn operon in Halobacterium salinarum and the canonical rrnA operon in Haloarcula marismortui. The proportion of modified nucleotides in tRNA can approach 50% or more. The two most frequent modifications in RNA are ribose methylation and the pseudouridylation. In an attempt to distinguish between these two alternatives, actinomycin D was used to inhibit transcription and Northern hybridization was used to follow the decay of the various mRNA fragments. There was a strong correlation between fragment length and stability: the shorter the fragment, the longer the half-life. This correlation was used to argue that the transcript fragments are generated by endonucleotic cleavage rather than premature transcription termination, and that the distal sequences released following cleavage are selectively degraded.
Maturation of precursor tRNA in Archaea. Precursor tRNAs are transcribed with 5ʹ-leader and 3ʹ-trailer sequences that have to be removed to yield a functional tRNA. Processing at the 5ʹ end is performed by the ubiquitous enzyme RNase P. The tRNA 3ʹ-end maturation is catalyzed by the endonuclease tRNase Z; after this the 3ʹ-terminal CCA sequence is added by the tRNA nucleotidyltransferase. Some archaeal tRNA precursors contain introns that are removed by the splicing endonuclease, which recognizes the bulge-helix-bulge (BHB) structural motif that forms between the exon/intron and intron/exon boundaries. The two halves of the tRNA that are generated by intron excision are joined by a tRNA ligase activity that has not yet been identified or characterized. In addition to nucleolytic processing, numerous nucleosides in the tRNA are subjected to modification. After all these processing and modification steps, the tRNA is ready for aminoacylation. The order of processing events is not known, and the scheme depicted is not necessarily what occurs in vivo.
Structure of the rRNA operons and processing of the ribosomal RNA precursor. (a) The structure of a typical rRNA operon from Euryarchaeota is shown. 16S, 23S, 5S rRNA, tRNAAla, and tRNACys genes are represented by solid black boxes; inverted repeats flanking the 16S and 23S genes are indicated by hatched boxes. Sequences are not drawn to scale. (b) The rRNA operon is transcribed as a multicistronic precursor molecule and is cleaved at numerous sites by a variety of endonucleases (indicated by black arrows and scissors). The splicing endonuclease (SE) cleaves at the BHB motifs to excise the precursor 16S and precursor 23S rRNA sequences, and RNase P and tRNase Z remove tRNAAla from the internal transcribed spacer region ( 47 ). The activities responsible for maturation at the 5ʹ and 3ʹ ends of the 16S and 23S rRNAs have not been identified or characterized. There is some evidence to suggest that the 5S sequence is excised from the primary transcript as a precursor (indicated by black arrows upstream and downstream from the 5S rRNA) and trimmed by a few nucleotides at both the 5ʹ and 3ʹ ends to generate the mature 5S sequence ( 47 ). The cleavage site in tRNase Z remove tRNAAla from the internal transcribed arrow and 3ʹ) was mapped as a 3ʹ-end site and there was no corresponding 5ʹ-end site at the same position ( 47 ). Thus this particular 3ʹ end is either generated by exonucleolytic trimming from the 3ʹ end of the tRNA, or it is generated by an endonucleolytic cleavage in the anticodon loop and the other product containing the tRNA 3ʹ half is degraded. The processing sites for the tRNACys have not been mapped experimentally, but tRNACys 5ʹ and 3ʹ processing is very likely to be performed by RNase P and tRNase Z, respectively. The 5ʹ-ETS is located upstream of the 16S rRNA, ITS1 is located between the 16S and the 23S rRNA, ITS2 is located between the 23S and the 5S rRNA, and the 3ʹ-ETS is downstream of the 5S rRNA.
Principles of mRNA decay in Bacteria and Eucarya and model for mRNA decay by exosomes in Archaea. The addition of poly(A) destabilizes transcripts in Bacteria, and in the nucleus of Eucarya. Exonucleolytic activity of the exosome has been demonstrated, but the involvement of endonucleases in mRNA turnover is still uncertain. Differential stabilities of mRNA segments can control protein levels in Bacteria and Archaea. Differently shaded bars indicate different ORFs in Bacteria and Archaea, and introns and exons in Eucarya. Short black bars indicate endonucleolytic cleavage sites in bacterial mRNA. En-doribonucleases are symbolized by scissors, exoribonucleases by “pacmen,” the exosome by multiple “pacmen.” The black hair-pin structures represent stabilizing mRNA secondary structures.
RNA-degrading protein complexes in members of the three domains of life. The degradosome of gram-negative bacteria is organized around endoribonuclease E. Association of RNase E with an exoribonuclease and one or more helicases seems to be conserved. The exoribonuclease PNPase consists of a trimer, each of which contains two RNase PH domains, one KH- and one S1 RNA-binding domain. A similar hexameric structure was found for the central ring of the exosome in Archaea and Eucarya, which is composed of six subunits with RNase PH domain. The exosome subunits Rrp4 and Rrp40 show the typical KH- and S1 RNA-binding domains of hydrolytic exoribonucleases. Rrp44 and Csl4, and RNase R comprise the S1 DNA-binding domain. In the Sulfolobus exosome core, Rrp41 is catalytically active while Rrp42 is not but contributes to the structuring of the Rrp41 active site.