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Escherichia coli K-12 substr. MG1655 Enzyme: RNase T



Gene: rnt Accession Numbers: EG11547 (EcoCyc), b1652, ECK1648

Regulation Summary Diagram: ?

Subunit composition of RNase T = [Rnt]2

Summary:
RNase T is a 3'-5' exonuclease responsible for 3' trimming of many stable RNAs, including tRNAs. It is also capable of degrading single-stranded DNA, though no in vivo substrates have been identified.

RNase T participates in tRNA maturation and can compensate for the lack of RNases II, D, BN, T and PH, which also carry out tRNA maturation [Reuven93]. tRNA in mutants lacking RNase T activity is slightly less amenable to aminoacylation, and rnt mutants lacking other tRNA-maturation RNases grow slowly and recover poorly from starvation [Padmanabha91]. However, reduced RNase T activity eliminates the slow growth and accumulation of defective tRNA seen in Cca mutants that lack tRNA nucleotidyltransferase activity [Deutscher85a].

RNase T is one of several RNases that can carry out 3' exoribonucleolytic cleavage of small, stable RNAs such as M1, 10Sa, 6S and 4.5S RNA. It appears to be able to cleave more closely to predicted double-stranded secondary structure than other 3' exoribonucleases [Li98]. RNase T is required for 5S RNA maturation, with its absence resulting in two extra 3' nucleotides that can be removed with purified RNase T in vitro [Li95]. It is also involved in maturation of 23S rRNA, being required for removal of the last few 3' precursor nucleotides [Li99].

RNase T also has a 3'-5' exonuclease activity against single-stranded DNA, with a much higher substrate affinity for DNA than RNA [Viswanathan98]. This may explain why rnt is a high-copy suppressor of UV sensitivity in strains lacking the DNA-specific exonucleases RecJ, Exo I and Exo VII [Viswanathan99]. As in RNA cleavage, RNase T cleaves even immediately next to double-stranded DNA [Zuo99].

RNase T is active as a dimer; its dimerization depends on Cys-168 and the RNase T carboxy-terminus [Li96, Zuo02b]. RNase T has three DEDD motifs that are required for catalysis and three NBS motifs that are required for substrate binding [Zuo02b]. The substrate binding face of one Rnt monomer in a dimer interacts with the catalytic face of the other, and vice-versa, allowing the catalytic activity to reach the substrate [Zuo02a].

Though it may bind up to ten nucleotides in its active site, the substrate specificity of RNase T is largely controlled by the final four. 3'-terminal C and CC drastically limit substrate cleavage [Zuo02]. Perhaps as a consequence, tRNA-CCA is readily degraded, whereas tRNA-CC is not [Deutscher84].

A strain lacking RNases II, D, BN, T and PH is inviable, but any one of these exoribonucleases can restore viability, with RNase T being the most effective in that capacity [Kelly92a].

An rnt mutant has slightly reduced ability to act as a recipient in cell-to-cell transfer of a plasmid [Kurono12].

Gene Citations: [Huang92]

Locations: cytosol

Map Position: [1,726,371 -> 1,727,018] (37.21 centisomes)
Length: 648 bp / 215 aa

Molecular Weight of Polypeptide: 23.523 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0005523 , CGSC:32271 , DIP:DIP-10734N , EchoBASE:EB1509 , EcoGene:EG11547 , EcoliWiki:b1652 , Mint:MINT-1248458 , ModBase:P30014 , OU-Microarray:b1652 , PortEco:rnt , PR:PRO_000023800 , Pride:P30014 , Protein Model Portal:P30014 , RefSeq:NP_416169 , RegulonDB:EG11547 , SMR:P30014 , String:511145.b1652 , Swiss-Model:P30014 , UniProt:P30014

Relationship Links: InterPro:IN-FAMILY:IPR005987 , InterPro:IN-FAMILY:IPR006055 , InterPro:IN-FAMILY:IPR012337 , InterPro:IN-FAMILY:IPR013520 , PDB:Structure:2IS3 , PDB:Structure:3NGY , PDB:Structure:3NGZ , PDB:Structure:3NH0 , PDB:Structure:3NH1 , PDB:Structure:3NH2 , PDB:Structure:3V9S , PDB:Structure:3V9U , PDB:Structure:3V9W , PDB:Structure:3V9X , PDB:Structure:3V9Z , PDB:Structure:3VA0 , PDB:Structure:3VA3 , Pfam:IN-FAMILY:PF00929 , Smart:IN-FAMILY:SM00479

Gene-Reaction Schematic: ?

GO Terms:

Biological Process: GO:0042780 - tRNA 3'-end processing Inferred from experiment [Reuven93]
GO:0090305 - nucleic acid phosphodiester bond hydrolysis Inferred by computational analysis Inferred from experiment [Li94a, UniProtGOA11a, GOA01]
GO:0006396 - RNA processing Inferred by computational analysis [GOA06, GOA01]
GO:0008033 - tRNA processing Inferred by computational analysis [UniProtGOA11a]
GO:0090501 - RNA phosphodiester bond hydrolysis Inferred by computational analysis [GOA01]
GO:0090503 - RNA phosphodiester bond hydrolysis, exonucleolytic Inferred by computational analysis [GOA06]
Molecular Function: GO:0008408 - 3'-5' exonuclease activity Inferred from experiment [Li94a]
GO:0000287 - magnesium ion binding Inferred by computational analysis [GOA06]
GO:0003676 - nucleic acid binding Inferred by computational analysis [GOA01]
GO:0004518 - nuclease activity Inferred by computational analysis [UniProtGOA11a]
GO:0004527 - exonuclease activity Inferred by computational analysis [UniProtGOA11a, GOA01]
GO:0004540 - ribonuclease activity Inferred by computational analysis [GOA01]
GO:0016787 - hydrolase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016896 - exoribonuclease activity, producing 5'-phosphomonoesters Inferred by computational analysis [GOA06]
GO:0046872 - metal ion binding Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, LopezCampistrou05]

MultiFun Terms: information transfer DNA related DNA degradation
information transfer RNA related RNA degradation
metabolism degradation of macromolecules DNA
metabolism degradation of macromolecules RNA

Essentiality data for rnt knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 1]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 2]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 1]

Credits:
Revised 25-May-2011 by Brito D


Enzymatic reaction of: RNase T exonuclease

a tRNA precursor with a short 3' extension <=> an uncharged tRNA + n a nucleoside 5'-monophosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: tRNA processing


Enzymatic reaction of: RNase T exonuclease

a tRNA precursor with a 5' extension and a short 3' extension <=> a tRNA precursor with a 5' extension + a ribonucleoside 5'-monophosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the Enzyme Commission system.

The reaction is favored in the direction shown.

In Pathways: tRNA processing


Enzymatic reaction of: RNase T exonuclease

EC Number: 3.1.13.-

RNA + n H2O <=> n a nucleoside 5'-monophosphate

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Summary:
The pH optimum is in the 8-9 range [Deutscher84].

Cofactors or Prosthetic Groups: Co2+ [Deutscher84], Mn2+ [Deutscher84], Mg2+ [Deutscher85]

pH(opt): 8.5 [Deutscher84]


Sequence Features

Feature Class Location Citations Comment
Conserved-Region 13 -> 15
[Zuo02b]
NBS1 motif, with conserved residues Arg-13 and Arg-15. Required for substrate binding.
Conserved-Region 19 -> 194
[UniProt09]
UniProt: Exonuclease;
Conserved-Region 23 -> 25
[Zuo02b]
Exo I DEDD exonuclease motif, with conserved residues Asp-23 and Glu-25. Required for catalysis.
Conserved-Region 108 -> 114
[Zuo02b]
NBS1 motif number two (NBS2), with conserved residues Lys-108 and Arg-114. Required for substrate binding.
Conserved-Region 120 -> 125
[Zuo02b]
Exo II DEDD exonuclease motif, with conserved residues His-120 and Asp-125. Required for catalysis.
Conserved-Region 139 -> 140
[Zuo02b]
NBS1 motif number three (NBS3), with conserved residues Lys-139 and Arg-140. Required for substrate binding.
Conserved-Region 181 -> 186
[Zuo02b]
Exo III DEDD exonuclease motif, with conserved residues His-181 and Asp-186. Required for catalysis.


Gene Local Context (not to scale): ?

Transcription Units:

Notes:

History:
10/20/97 Gene b1652 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11547; confirmed by SwissProt match.


References

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Deutscher84: Deutscher MP, Marlor CW, Zaniewski R (1984). "Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA." Proc Natl Acad Sci U S A 81(14);4290-3. PMID: 6379642

Deutscher85: Deutscher MP, Marlor CW (1985). "Purification and characterization of Escherichia coli RNase T." J Biol Chem 260(11);7067-71. PMID: 3888994

Deutscher85a: Deutscher MP, Marlor CW, Zaniewski R (1985). "RNase T is responsible for the end-turnover of tRNA in Escherichia coli." Proc Natl Acad Sci U S A 82(19);6427-30. PMID: 2413440

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

GOA01: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

GOA06: GOA, SIB (2006). "Electronic Gene Ontology annotations created by transferring manual GO annotations between orthologous microbial proteins."

Huang92: Huang S, Deutscher MP (1992). "Sequence and transcriptional analysis of the Escherichia coli rnt gene encoding RNase T." J Biol Chem 267(35);25609-13. PMID: 1460056

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Kelly92a: Kelly KO, Deutscher MP (1992). "The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli." J Bacteriol 174(20);6682-4. PMID: 1400219

Kurono12: Kurono N, Matsuda A, Etchuya R, Sobue R, Sasaki Y, Ito M, Ando T, Maeda S (2012). "Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells." Biochem Biophys Res Commun 421(1);119-23. PMID: 22497891

Li94a: Li Z, Deutscher MP (1994). "The role of individual exoribonucleases in processing at the 3' end of Escherichia coli tRNA precursors." J Biol Chem 269(8);6064-71. PMID: 7509797

Li95: Li Z, Deutscher MP (1995). "The tRNA processing enzyme RNase T is essential for maturation of 5S RNA." Proc Natl Acad Sci U S A 92(15);6883-6. PMID: 7542780

Li96: Li Z, Zhan L, Deutscher MP (1996). "Escherichia coli RNase T functions in vivo as a dimer dependent on cysteine 168." J Biol Chem 271(2);1133-7. PMID: 8557641

Li98: Li Z, Pandit S, Deutscher MP (1998). "3' exoribonucleolytic trimming is a common feature of the maturation of small, stable RNAs in Escherichia coli." Proc Natl Acad Sci U S A 95(6);2856-61. PMID: 9501180

Li99: Li Z, Pandit S, Deutscher MP (1999). "Maturation of 23S ribosomal RNA requires the exoribonuclease RNase T." RNA 5(1);139-46. PMID: 9917073

LopezCampistrou05: Lopez-Campistrous A, Semchuk P, Burke L, Palmer-Stone T, Brokx SJ, Broderick G, Bottorff D, Bolch S, Weiner JH, Ellison MJ (2005). "Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth." Mol Cell Proteomics 4(8);1205-9. PMID: 15911532

Padmanabha91: Padmanabha KP, Deutscher MP (1991). "RNase T affects Escherichia coli growth and recovery from metabolic stress." J Bacteriol 173(4);1376-81. PMID: 1704882

Reuven93: Reuven NB, Deutscher MP (1993). "Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo." FASEB J 7(1);143-8. PMID: 8422961

UniProt09: UniProt Consortium (2009). "UniProt version 15.8 released on 2009-10-01 00:00:00." Database.

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

Viswanathan98: Viswanathan M, Dower KW, Lovett ST (1998). "Identification of a potent DNase activity associated with RNase T of Escherichia coli." J Biol Chem 273(52);35126-31. PMID: 9857048

Viswanathan99: Viswanathan M, Lanjuin A, Lovett ST (1999). "Identification of RNase T as a high-copy suppressor of the UV sensitivity associated with single-strand DNA exonuclease deficiency in Escherichia coli." Genetics 151(3);929-34. PMID: 10049912

Zuo02: Zuo Y, Deutscher MP (2002). "The physiological role of RNase T can be explained by its unusual substrate specificity." J Biol Chem 277(33);29654-61. PMID: 12050169

Zuo02a: Zuo Y, Deutscher MP (2002). "Mechanism of action of RNase T. II. A structural and functional model of the enzyme." J Biol Chem 277(51);50160-4. PMID: 12364333

Zuo02b: Zuo Y, Deutscher MP (2002). "Mechanism of action of RNase T. I. Identification of residues required for catalysis, substrate binding, and dimerization." J Biol Chem 277(51);50155-9. PMID: 12364334

Zuo99: Zuo Y, Deutscher MP (1999). "The DNase activity of RNase T and its application to DNA cloning." Nucleic Acids Res 27(20);4077-82. PMID: 10497273

Other References Related to Gene Regulation

Reuven95: Reuven NB, Koonin EV, Rudd KE, Deutscher MP (1995). "The gene for the longest known Escherichia coli protein is a member of helicase superfamily II." J Bacteriol 1995;177(19);5393-400. PMID: 7559321


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 18.5 on Fri Dec 19, 2014, biocyc14.