Organization and nucleotide sequence of a new ribosomal operon in Escherichia coli containing the genes for ribosomal protein S2 and elongation factor Ts

An, Gynheung; Bendiak, Dirk S.; Mamelak, Linda; Friesen, James D.

doi:10.1093/nar/9.16.4163

Cited by 104 publications

(36 citation statements)

References 29 publications

(30 reference statements)

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“…5). Such an attenuation mechanism had already been postulated by An et al (1981) for the rpsB-tsf operon in E. coli on the basis of a long but imperfect inverted repeat in the intergenic region. In the intergenic region of the S. coelicolor rpsB-tsf operon we see a perfect inverted repeat consisting of two 16 bp elements, possibly allowing Rhoindependent termination of the RNA polymerase.…”

Section: Discussionmentioning

confidence: 80%

Evidence that a single EF-Ts suffices for the recycling of multiple and divergent EF-Tu species in Streptomyces coelicolor A3(2) and Streptomyces ramocissimus The GenBank accession numbers for the sequences of the S. coelicolor rpsB–tsf operon and the S. ramocissimus tsf gene determined in this work are AF034101 and AF130345, respectively.

1999

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The tsf genes from Streptomyces coelicolor A3(2) and Streptomyces ramocissimus , encoding the guanine-nucleotide exchange factor EF-Ts, were cloned and sequenced. Streptomycetes have multiple and highly divergent EFTu species, with EF-Tu1 and EF-Tu3 showing only about 65 % amino acid sequence identity, and yet these can apparently interact with a single EF-Ts species. tsf lies in an operon with rpsB, which encodes ribosomal protein S2. The amino acid sequence of S2 from S. coelicolor differs from most other bacterial S2 homologues in having a C-terminal extension of 70 aa residues with a highly repetitive organization, the function of which is unknown. Transcription analysis of the rpsB-tsf operon of S. coelicolor by promoter probing, nuclease S1 mapping and Northern blotting revealed that the genes give rise to a bicistronic transcript from a single promoter upstream of rpsB. An attenuator was identified in the rpsB-tsf intergenic region ; it results in an approximately 2 :1 ratio of rpsB vs tsf transcripts. Although tuf1, encoding the major EF-Tu, is located in the rpsL ribosomal protein operon, an additional promoter in the fus-tuf1 intergenic region leads to a significant excess of EFTu over ribosomes. Most amino acid residues known from the Escherichia coli crystal structure of the EF-Tu EF-Ts complex to be directly involved in interaction between the two elongation factors are conserved between E. coli and Streptomyces. However, whenever interaction residues in the EF-Tu moiety show divergence among Streptomyces EF-Tu1, EF-Tu2 and EF-Tu3, the single Streptomyces EF-Ts exhibits compensatory substitutions of the corresponding residues. These apparently enable productive interaction to occur with all three EF-Tus.

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Section: Discussionmentioning

confidence: 80%

Evidence that a single EF-Ts suffices for the recycling of multiple and divergent EF-Tu species in Streptomyces coelicolor A3(2) and Streptomyces ramocissimus The GenBank accession numbers for the sequences of the S. coelicolor rpsB–tsf operon and the S. ramocissimus tsf gene determined in this work are AF034101 and AF130345, respectively.

1999

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“…The hUGDH gene is flanked by unique EcoRI and HindIII restriction sites that render the gene portable to various E. coli expression vectors. Second, the codon usage of the resulting hUGDH gene was modified to include those triplets that are utilized in highly expressed E. coli genes (35,36) while retaining the largest possible number of unique restriction sites. In some cases, suboptimal codons were used either to allow the inclusion of unique restriction sites or to preclude redundant sites.…”

Section: Materials-udp-glucose Nadmentioning

confidence: 99%

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Huh

Yoon²,

Lee

et al. 2004

Journal of Biological Chemistry

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UDP-glucose dehydrogenase (UGDH) is (6, 7) show that the reaction of UGDH involves two successive oxidations to convert the 6Ј-hydroxyl of UDP-glucose to a carboxylate, together with the reduction of 2 molecules of NAD ϩ to NADH. In animals, it constitutes the unique pathway for glucuronate formation (8). Because glucuronate is a component of glycosaminoglycans (GAGs), the mutation inactivation of UGDH (sugarless) abolishes GAG assembly and, consequently, abolishes GAG-dependent growth factor signaling (9 -11). The primary structure of the mammalian enzyme was obtained from the protein sequence of bovine UGDH (12). The human gene was also recently cloned and assigned to chromosome 4p15.1 (13). It contains 12 exons, extends over 26 kb, and has one major transcription start site (14).GAG chains of proteoglycans and hyaluronan are ubiquitous components of extracellular matrix and pericellular spaces. There is a growing body of information on the implication of GAGs in cell behavior, including signal transduction, cell proliferation, spreading, migration, and cancer growth and metastasis (15-17). GAG synthesis is influenced by cytokines and growth factors. Transforming growth factor-␤ is the most potent stimulator of proteoglycan and GAG synthesis, including that of hyaluronan. Its action, however, depends on the cell type (18). The synthesis of GAGs is also modulated by oxygen cell status. Hypoxic endothelial cells and lung fibroblasts enhanced the heparan sulfate/chondroitin sulfate ratio, which led to an increase of basic fibroblast growth factor reactivity on the cell surface (19,20). It was also shown that the level of intracellular UDP-glucuronate could influence GAG synthesis (8). Interestingly, the human UGDH was shown to be an early response gene after interleukin-1 treatment of ocular fibroblasts (21), as well as an early androgen response gene in breast cancer (22).Although a clone of human UGDH has been reported (13, 15), specific catalytic residues are not yet available. Therefore, further characterization of the active sites of human UGDH is needed to elucidate the physiological nature of the UGDH. In the present study, we have identified an NAD ϩ -binding site using photoaffinity labeling and cassette mutagenesis to gain a deeper insight into the structural basis of human UGDH. For this study, a 1509-base pair gene that encodes human UGDH has been chemically synthesized and expressed in Escherichia coli as a soluble protein. Identification of the nucleotide-binding sites of a variety of proteins has been advanced by the use of nucleotide photoaffinity analogues that selectively insert * This work was supported by Grant 03-PJ1-PG3-20900-0047 from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Korea (to S.-W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

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“…3. Amino acid sequence of EF-Ts as derived from the gene sequence [22]. Trypsin cleavage occurs on the C-terminal side of the residues marked with asterisks.…”

Section: Z Crystallization and X-ray Analysis Of The C-terminal Fragmentmentioning

confidence: 99%

“…However, a recent study by Hwang et al [21] indicates that also residues 154-199 located in domain I of EF-Tu are involved in EF-Ts binding. Very little structural information about EF-Ts is available, other than the nucleotide sequence of the EF-Ts encoding gene tsf [22]. More information concerning the EF-Tu:EF-Ts interaction could come from Xray analysis of EF-Ts and the EF-Tu: EF-Ts complex.…”

Section: Introductionmentioning

confidence: 99%

Analysis and crystallization of a 25 kDa C‐terminal fragment of cloned elongation factor Ts fromEscherichia coli

et al. 1995

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A 25 kDa C-terminal tryptic fragment of elongation factor "Is has been purified to homogeneity. Experimental evidence suggests that the 25 kDa C-terminal and the 5.3 kDa N-terminal fragments are structurally independent domains. The N-terminal fragment is shown to be essential for the nucleotide exchange activity. Crystals of the C-terminal fragment belong to space group P2 or P21. The diffraction pattern shows a pronounced pseudo-C2 symmetry at low resolution. This pseudo symmetry increases when the crystals are irradiated with X-rays for a few hours.

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Organization and nucleotide sequence of a new ribosomal operon in Escherichia coli containing the genes for ribosomal protein S2 and elongation factor Ts

Cited by 104 publications

References 29 publications

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

Analysis and crystallization of a 25 kDa C‐terminal fragment of cloned elongation factor Ts fromEscherichia coli

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