The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Abstract

RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2 -O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5 -monophosphate RNA termini but has an inherent mode of cleavage in the 3 to 5 direction. Target sequences made uncleavable by the introduction of 2 -O-methylmodified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5 to, but not 3 to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3 to 5 ''scanning'' mechanism and imply that anchoring of the enzyme to the 5 -monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.has a demonstrated role in the processing of ribosomal RNA (1, 2), the chemical degradation of bulk cellular RNA (3-7), the decay of specific regulatory, messenger, and structural RNAs (for recent reviews, see refs. 8 and 9), the control of plasmid DNA replication (10), and the removal of poly(A) tails from transcripts (11,12). RNase E cleaves preferentially at specific sites in single-strand RNA segments rich in AϩU nucleotides (13)(14)(15)(16)). An inherent mode of action involving entry of RNase E at the 5Ј end of substrates followed by a 5Ј to 3Ј wave of cleavages has been inferred from the greater in vivo stability of fragments at the 3Ј ends of certain RNA substrates (17-20) together with evidence that (i) the enzyme prefers a free 5Ј end for endonucleolytic cleavage (18,20), (ii) RNase E degradation efficiency is affected by 5Ј-phosphorylation in vivo (10) and in vitro (19,20), and (iii) 5Ј regions of secondary structure can impede cleavages in vivo (21). However, RNA degradation in vivo also can occur in the opposite direction (e.g., ref. 22).As RNase E cleavage of complex substrates potentially can be affected by differences in the affinity of the enzyme for target sites having different sequences (15, 16), by secondary structure (23), and in vivo also by the attachment of ribosomes (24-26) and͞or RNA-binding proteins (27), conclusions about the inherent mode of action of RNase E from studies of long natural substrates may be problematical. We wished to elucidate the mechanism of target-site selection by RNase E in the absence of these confounding factors; to do this, we designed, and chemically synthesized, oligoribonucleotide substrates that contain repeats of identical target sequences and are devoid of regions able to engage in base paring. We found that the RNase E catalytic...

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

“…In light of the result with the deoxy-2Ј-fluorouridine-substituted substrate, this finding suggests that RNaseE hydrolysis is hampered by a bulky group at this particular position. Although 2Ј-O-methylcontaining oligonucleotides have been shown previously not to be cleaved by RNaseE, the cause could not be linked to a specific position because more than one substitution had been incorporated (42). DISCUSSION A number of factors have been shown to influence hydrolysis of RNA by E. coli RNaseE such as the sequence of hydrolysis sites, the secondary structure and 5Ј phosphorylation status of the substrate, and domains within the C-terminal half of the enzyme.…”

Retention of Core Catalytic Functions by a Conserved Minimal Ribonuclease E Peptide That Lacks the Domain Required for Tetramer Formation

“…of RNase E and RNase G-N-Rne has been employed previously for investigation of substrate specificity (14,17), mode of action (28), and inhibition by other cellular proteins (34). To identify the minimal domain that retained activity, additional constructs for expression of N-Rne fragments with deletions from either the amino or the carboxyl terminus were made (Fig.…”

“…The BR30M RNA substrate and 50 ng of protein were used for each assay. Only E. coli RNase G, which cleaves substrate randomly without directionality, can cleave BR30M at the 5Ј end from the modified nucleotides (5 and 6 nt) (25,28).…”

“…RNase E cleavage specificity and mode of action (28). BR30M contains three repeats of a sequence that normally is susceptible to RNase E cleavage.…”

“…Although all members of the RNase E/G family that have been experimentally studied cleave their substrates in A ϩ U-rich regions, the substrate sites cleaved by RNase G and RNase E are not identical (9,25). Moreover, despite extensive homology between the catalytic domains of all members of the RNase E/G family, biochemical studies indicate that the RNase E of E. coli can degrade substrates quasi-processively in a 3Ј to 5Ј scanning mode, whereas RNase G from the same organism has a distributive mode of action (28).…”