rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes

Jacob Crandall, Milagros Rodriguez-Lopez, Michael Pfeiffer, Bailey Mortensen, Allen Buskirk

Research output: Contribution to journalArticle

Abstract

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3′ end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).

Original languageEnglish (US)
Pages (from-to)553-559
Number of pages7
JournalJournal of Bacteriology
Volume192
Issue number2
DOIs
StatePublished - Jan 2010
Externally publishedYes

Fingerprint

Transfer RNA
Ribosomes
Messenger RNA
Mutation
Codon
5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide
Peptides
Terminator Codon
Genetic Selection
Quality Control
Fingers
Proteins
Cell Death
Bacteria

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes. / Crandall, Jacob; Rodriguez-Lopez, Milagros; Pfeiffer, Michael; Mortensen, Bailey; Buskirk, Allen.

In: Journal of Bacteriology, Vol. 192, No. 2, 01.2010, p. 553-559.

Research output: Contribution to journalArticle

Crandall, J, Rodriguez-Lopez, M, Pfeiffer, M, Mortensen, B & Buskirk, A 2010, 'rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes', Journal of Bacteriology, vol. 192, no. 2, pp. 553-559. https://doi.org/10.1128/JB.01178-09
Crandall, Jacob ; Rodriguez-Lopez, Milagros ; Pfeiffer, Michael ; Mortensen, Bailey ; Buskirk, Allen. / rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes. In: Journal of Bacteriology. 2010 ; Vol. 192, No. 2. pp. 553-559.
@article{d66ba5f1fce441568f36f70fb67b5f41,
title = "rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes",
abstract = "In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3′ end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).",
author = "Jacob Crandall and Milagros Rodriguez-Lopez and Michael Pfeiffer and Bailey Mortensen and Allen Buskirk",
year = "2010",
month = "1",
doi = "10.1128/JB.01178-09",
language = "English (US)",
volume = "192",
pages = "553--559",
journal = "Journal of Bacteriology",
issn = "0021-9193",
publisher = "American Society for Microbiology",
number = "2",

}

TY - JOUR

T1 - rRNA mutations that inhibit transfer-messenger RNA activity on stalled ribosomes

AU - Crandall, Jacob

AU - Rodriguez-Lopez, Milagros

AU - Pfeiffer, Michael

AU - Mortensen, Bailey

AU - Buskirk, Allen

PY - 2010/1

Y1 - 2010/1

N2 - In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3′ end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).

AB - In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner, SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated rRNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We found that deletion of A1150 in the 16S rRNA blocked tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowered tagging levels at Pro-opal and rare Arg codons, but not at the 3′ end of an mRNA lacking a stop codon. We concluded that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging. C889 is found in the A-site finger, where it interacts with the S13 protein in the small subunit (forming intersubunit bridge B1a).

UR - http://www.scopus.com/inward/record.url?scp=73649149025&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73649149025&partnerID=8YFLogxK

U2 - 10.1128/JB.01178-09

DO - 10.1128/JB.01178-09

M3 - Article

C2 - 19897649

AN - SCOPUS:73649149025

VL - 192

SP - 553

EP - 559

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 2

ER -