Escherichia coli Poly(A)-binding Proteins That Interact with Components of Degradosomes or Impede RNA Decay Mediated by Polynucleotide Phosphorylase and RNase E

Abstract

The multifunctional ribonuclease RNase E and the 3-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3 ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 M salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3 to 5 exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5 ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.

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

“…The ability 5Ј-phosphorylation to influence multiple types of transactions occurring at 3Ј ends supports the notion that the termini of completed E. coli transcripts may be brought into proximity by degradosome component proteins, as suggested (11,37,(58)(59)(60); for review, see ref. 61).…”

Three Amino Acids in Escherichia coli CspE Surface-exposed Aromatic Patch Are Critical for Nucleic Acid Melting Activity Leading to Transcription Antitermination and Cold Acclimation of Cells

“…We show that in addition to His 32 , Phe 17 and Phe 30 residues are also important for nucleic acid melting by CspE. On the other hand, substitutions of peripheral aromatic patch amino acid residues, Trp 10 , Phe 19 , and Phe 33 , have no effect on nucleic acid melting. Thus, the centrally located aromatic patch triad of Phe 17 , Phe 30 , and His 32 in CspE appears to initiate nucleic acid melting, which is then extended further to the periphery of the patch.…”

RNA remodeling and gene regulation by cold shock proteins

“…The proteins carrying substitutions of these amino acids with Arg fully retain their nucleic acid binding activity, but lose the ability to cause transcription antitermination and cold acclimation of cells as they lack the melting activity. 40,77 DNA microarray analysis of the cold shock response of the wild-type to cold-shock, stationary phase, nutrient starvation and freezing, 47,48 antibiotic biosynthesis, 49 UV sensitivity, 50 regulation of expression of proteins responding to osmotic stress, oxidative stress and stationary phase (UspA, OsmY, Dps, ProP and KatG 51 ), camphor resistance and chromosome condensation, 52,53 downregulation of λ Q-mediated transcription antitermination, 54 downregulation of poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase, 55 inhibition of DNA replication, 56 and tolerance to solvents such as toluene. 57 The cold-shock induction of CspA and its homologs was studied in detail and was shown to occur at the levels of transcription, mRNA stability and translation.…”