A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Lisa M. Sharpe Elles; Michael T. Sykes; James R. Williamson; Olke C. Uhlenbeck

doi:10.1093/nar/gkp711

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Lisa M. Sharpe Elles, Michael T. Sykes, James R. Williamson, Olke C. Uhlenbeck^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

57 Scopus citations

Abstract

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg²⁺ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

Original language	English (US)
Pages (from-to)	6503-6514
Number of pages	12
Journal	Nucleic acids research
Volume	37
Issue number	19
DOIs	https://doi.org/10.1093/nar/gkp711
State	Published - 2009

Funding

National Institute of General Medical Sciences Award Number (F32GM083510 to M.T.S.); the National Institutes of Health (Grant R37-GM53757 to J.R.W.); and the National Institutes of Health (Grant R01-GM60268-08 to O.C.U.). Funding for the open access charge: Discretionary funds from Northwestern University (O.C.U.).

ASJC Scopus subject areas

Genetics

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title = "A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit",

abstract = "Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.",

author = "{Sharpe Elles}, {Lisa M.} and Sykes, {Michael T.} and Williamson, {James R.} and Uhlenbeck, {Olke C.}",

note = "Funding Information: National Institute of General Medical Sciences Award Number (F32GM083510 to M.T.S.); the National Institutes of Health (Grant R37-GM53757 to J.R.W.); and the National Institutes of Health (Grant R01-GM60268-08 to O.C.U.). Funding for the open access charge: Discretionary funds from Northwestern University (O.C.U.).",

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AU - Sykes, Michael T.

AU - Williamson, James R.

AU - Uhlenbeck, Olke C.

N1 - Funding Information: National Institute of General Medical Sciences Award Number (F32GM083510 to M.T.S.); the National Institutes of Health (Grant R37-GM53757 to J.R.W.); and the National Institutes of Health (Grant R01-GM60268-08 to O.C.U.). Funding for the open access charge: Discretionary funds from Northwestern University (O.C.U.).

PY - 2009

Y1 - 2009

N2 - Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

AB - Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

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A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

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