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

2002 ◽  
Vol 99 (23) ◽  
pp. 14746-14751 ◽  
Author(s):  
Y. Feng ◽  
T. A. Vickers ◽  
S. N. Cohen
Keyword(s):  
Site Selection ◽  
Cleavage Site ◽  
Catalytic Domain ◽  
Rnase E

scholarly journals Activation of the Kexin from Schizosaccharomyces pombeRequires Internal Cleavage of Its Initially Cleaved Prosequence

1998 ◽  
Vol 18 (1) ◽  
pp. 400-408 ◽  
Author(s):  
Dale Powner ◽  
John Davey
Keyword(s):  
Cleavage Site ◽  
Secretory Pathway ◽  
Catalytic Domain ◽  
Activation Process ◽  
Processing Enzymes ◽  
Free Translation ◽  
A Cell ◽  
Internal Site ◽  
Primary Cleavage ◽  
Full Activation

ABSTRACT Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeastSchizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopuseggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombecells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.

scholarly journals Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

2018 ◽  
Author(s):  
Taylor B Updegrove ◽  
Andrew B Kouse ◽  
Katarzyna J Bandyra ◽  
Gisela Storz
Keyword(s):  
Small Rna ◽  
Cleavage Site ◽  
Differential Sensitivity ◽  
Rnase E ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Derivatives Of

AbstractIncreasing numbers of 3′UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3′ UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3′ UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3′ to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

scholarly journals Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover

Nature ◽  
2005 ◽  
Vol 437 (7062) ◽  
pp. 1187-1191 ◽  
Author(s):  
Anastasia J. Callaghan ◽  
Maria Jose Marcaida ◽  
Jonathan A. Stead ◽  
Kenneth J. McDowall ◽  
William G. Scott ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Rna Turnover ◽  
Rnase E

scholarly journals DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes

1999 ◽  
Vol 18 (9) ◽  
pp. 2638-2647 ◽  
Author(s):  
Pavel Janscak ◽  
Maria P. MacWilliams ◽  
Ursula Sandmeier ◽  
Valakunja Nagaraja ◽  
Thomas A. Bickle
Keyword(s):  
Site Selection ◽  
Cleavage Site ◽  
Restriction Enzymes ◽  
General Mechanism ◽  
Type I ◽  
Dna Translocation

scholarly journals RNase II binds to RNase E and modulates its endoribonucleolytic activity in the cyanobacterium Anabaena PCC 7120

2020 ◽  
Vol 48 (7) ◽  
pp. 3922-3934 ◽  
Author(s):  
Cong Zhou ◽  
Juyuan Zhang ◽  
Xinyu Hu ◽  
Changchang Li ◽  
Li Wang ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Bacterial Species ◽  
Rna Degradation ◽  
Rnase E ◽  
Anabaena Pcc 7120 ◽  
Cyanobacterium Anabaena ◽  
Rna Degradosome ◽  
Rnase Ii ◽  
Pcc 7120

Abstract In Escherichia coli, the endoribonuclease E (RNase E) can recruit several other ribonucleases and regulatory proteins via its noncatalytic domain to form an RNA degradosome that controls cellular RNA turnover. Similar RNA degradation complexes have been found in other bacteria; however, their compositions are varied among different bacterial species. In cyanobacteria, only the exoribonuclease PNPase was shown to bind to the noncatalytic domain of RNase E. Here, we showed that Alr1240, a member of the RNB family of exoribonucleases, could be co-isolated with RNase E from the lysate of the cyanobacterium Anabaena PCC 7120. Enzymatic analysis revealed that Alr1240 is an exoribonuclease II (RNase II), as it only degrades non-structured single-stranded RNA substrates. In contrast to known RNase E-interacting ribonucleases, which bind to the noncatalytic domain of RNase E, the Anabaena RNase II was shown to associate with the catalytic domain of RNase E. Using a strain in which RNase E and RNase II were tagged in situ with GFP and BFP, respectively, we showed that RNase E and RNase II form a compact complex in vivo by a fluorescence resonance energy transfer (FRET) assay. RNase E activity on several synthetic substrates was boosted in the presence of RNase II, suggesting that the activity of RNase E could be regulated by RNase II-RNase E interaction. To our knowledge, Anabaena RNase II is an unusual ribonuclease that interacts with the catalytic domain of RNase E, and it may represent a new type of RNA degradosome and a novel mechanism for regulating the activity of the RNA degradosome. As Anabaena RNase E interacts with RNase II and PNPase via different regions, it is very likely that the three ribonucleases form a large complex and cooperatively regulate RNA metabolism in the cell.

scholarly journals DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

2011 ◽  
Vol 39 (18) ◽  
pp. 8042-8051 ◽  
Author(s):  
Friedrich W. Schwarz ◽  
Kara van Aelst ◽  
Júlia Tóth ◽  
Ralf Seidel ◽  
Mark D. Szczelkun
Keyword(s):  
Site Selection ◽  
Dna Cleavage ◽  
Cleavage Site ◽  
Restriction Enzymes ◽  
Type Iii
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