A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits

T. Martin Schmeing, Amy C. Seila, Jeffrey L. Hansen, Betty Freeborn, Juliane K. Strauss-Soukup, Stephen A. Scaringe, Scott A. Strobel, Peter B. Moore, Thomas A. Steitz

Research output: Contribution to journalArticle

169 Citations (Scopus)

Abstract

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3′ OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

Original languageEnglish
Pages (from-to)225-230
Number of pages6
JournalNature Structural Biology
Volume9
Issue number3
DOIs
StatePublished - 2002

Fingerprint

Large Ribosome Subunits
Peptides
Crystals
Haloarcula marismortui
Peptidyl Transferases
Puromycin
Assays
Proteins
Methanol
Ethanol
Substrates
Alcohols
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Structural Biology
  • Genetics

Cite this

A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits. / Schmeing, T. Martin; Seila, Amy C.; Hansen, Jeffrey L.; Freeborn, Betty; Strauss-Soukup, Juliane K.; Scaringe, Stephen A.; Strobel, Scott A.; Moore, Peter B.; Steitz, Thomas A.

In: Nature Structural Biology, Vol. 9, No. 3, 2002, p. 225-230.

Research output: Contribution to journalArticle

Schmeing, TM, Seila, AC, Hansen, JL, Freeborn, B, Strauss-Soukup, JK, Scaringe, SA, Strobel, SA, Moore, PB & Steitz, TA 2002, 'A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits', Nature Structural Biology, vol. 9, no. 3, pp. 225-230. https://doi.org/10.1038/nsb758
Schmeing, T. Martin ; Seila, Amy C. ; Hansen, Jeffrey L. ; Freeborn, Betty ; Strauss-Soukup, Juliane K. ; Scaringe, Stephen A. ; Strobel, Scott A. ; Moore, Peter B. ; Steitz, Thomas A. / A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits. In: Nature Structural Biology. 2002 ; Vol. 9, No. 3. pp. 225-230.
@article{200bfc0c5c4f43ceb023e4fa5f688fbc,
title = "A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits",
abstract = "The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3′ OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.",
author = "Schmeing, {T. Martin} and Seila, {Amy C.} and Hansen, {Jeffrey L.} and Betty Freeborn and Strauss-Soukup, {Juliane K.} and Scaringe, {Stephen A.} and Strobel, {Scott A.} and Moore, {Peter B.} and Steitz, {Thomas A.}",
year = "2002",
doi = "10.1038/nsb758",
language = "English",
volume = "9",
pages = "225--230",
journal = "Nature Structural and Molecular Biology",
issn = "1545-9993",
publisher = "Nature Publishing Group",
number = "3",

}

TY - JOUR

T1 - A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits

AU - Schmeing, T. Martin

AU - Seila, Amy C.

AU - Hansen, Jeffrey L.

AU - Freeborn, Betty

AU - Strauss-Soukup, Juliane K.

AU - Scaringe, Stephen A.

AU - Strobel, Scott A.

AU - Moore, Peter B.

AU - Steitz, Thomas A.

PY - 2002

Y1 - 2002

N2 - The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3′ OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

AB - The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3′ OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.

UR - http://www.scopus.com/inward/record.url?scp=0036177483&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036177483&partnerID=8YFLogxK

U2 - 10.1038/nsb758

DO - 10.1038/nsb758

M3 - Article

VL - 9

SP - 225

EP - 230

JO - Nature Structural and Molecular Biology

JF - Nature Structural and Molecular Biology

SN - 1545-9993

IS - 3

ER -