The 5' end of the 18S rRNA can be positioned from within the mature rRNA.

In yeast, the 5' end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5' region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  E. Petfalski,et al.  Processing of the Precursors to Small Nucleolar RNAs and rRNAs Requires Common Components , 1998, Molecular and Cellular Biology.

[3]  C. Pleij,et al.  The central pseudoknot in 16S ribosomal RNA is needed for ribosome stability but is not essential for 30S initiation complex formation. , 1996, Nucleic acids research.

[4]  D. Tollervey,et al.  Yeast nucleoporin mutants are defective in pre-tRNA splicing , 1996, Molecular and cellular biology.

[5]  D. Tollervey,et al.  Two distinct recognition signals define the site of endonucleolytic cleavage at the 5′‐end of yeast 18S rRNA. , 1995, The EMBO journal.

[6]  E. Petfalski,et al.  The 5′ end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site. , 1994, The EMBO journal.

[7]  S. Liebman,et al.  An accuracy center in the ribosome conserved over 2 billion years. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Alksne,et al.  A novel cloning strategy reveals the gene for the yeast homologue to Escherichia coli ribosomal protein S12. , 1993, The Journal of biological chemistry.

[9]  H. Noller,et al.  A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly. , 1993, Genes & development.

[10]  C. Carles,et al.  Gene RRN4 in Saccharomyces cerevisiae encodes the A12.2 subunit of RNA polymerase I and is essential only at high temperatures , 1993, Molecular and cellular biology.

[11]  S. Liebman,et al.  The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein. , 1992, Genetics.

[12]  D. Tollervey,et al.  Identification and functional analysis of two U3 binding sites on yeast pre‐ribosomal RNA. , 1992, The EMBO journal.

[13]  R. Schiestl,et al.  Improved method for high efficiency transformation of intact yeast cells. , 1992, Nucleic acids research.

[14]  Y. Nogi,et al.  Synthesis of large rRNAs by RNA polymerase II in mutants of Saccharomyces cerevisiae defective in RNA polymerase I. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[15]  E. Otaka,et al.  Sequence and functional similarity between a yeast ribosomal protein and the Escherichia coli S5 ram protein , 1990, Molecular and cellular biology.

[16]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[17]  D. Tollervey.  A yeast small nuclear RNA is required for normal processing of pre‐ribosomal RNA. , 1987, The EMBO journal.

[18]  E. Petfalski,et al.  Recognition of cleavage site A(2) in the yeast pre-rRNA. , 1996, RNA.

[19]  Gerald R. Fink,et al.  Methods in Yeast Genetics: A Laboratory Course Manual , 1987 .