Biogenesis of small noncoding RNAs in escherichia coli

Abstract

Small noncoding RNAs, broadly defined as non-messenger RNAs with 50 to 500 nucleotides in size, are generated from primary transcripts through processing reactions by various ribonucleases (RNases) in Escherichia coli. Many cellular RNases are participated in this reaction. RNase E, one of the major endoribonucleases, is an endoribonuclease that is essential for cell viability by participating in both RNA processing and degradation. Therefore, substrate discrimination by RNase E is critical for its appropriate action within the cell. M1 RNA, the catalytic component of Escherichia coli RNase P, is derived by 3’ processing from pM1 RNA, a major transcript of the rnpB gene. To elucidate how RNase E discriminates substrate, in this study, cleavage of precursor M1 RNA variants containing various sequences at the rne-dependent site was analyzed with the N-terminal catalytic half of RNase E (NTH-RNase E). The wild-type GAUUU sequence was cleaved most efficiently among the variants. The cleavage efficiency of the UUUUU variant by NTH-RNase E was much less than the in vivo processing efficiency of that variant and for efficient cleavage the retention of single-strandedness at the rne-dependent site was required. Moreover, NTH-RNase E preferred G/C to A/U as the first nucleotide of the rne-dependent site. Kinetic analysis showed that NTH-RNase E displayed both higher $K_m$ and $k_{cat}$ values for more specific substrates. Although many small noncoding RNAs have been found, most of their biosynthetic pathways remain elusive. Biogenesis of 6S RNA that functions as a modulator of RNA polymerase ($σ^{70}$) activity has been unclear since it was detected 30 years ago. This study showed that there are two different precursors, the long and short ones, which are transcribed from the distal P2 and proximal P1 promoters, respectively. Transcription from the P2 promoter is $σ^S-dependent$, while that from the P1 promoter is not. Both precursors are processed to generate the 5’...