Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eukaryotic protein synthesis initiation factor 4E (eIF-4E) is a 25-kDa polypeptide that binds to the 7-methylguanosine-containing cap of mRNA and participates in the transfer of mRNA to the 40S ribosomal subunit, a step that is rate-limiting for protein synthesis under most cellular conditions. eIF-4E is the least abundant of the initiation factors, is present at approximately 10% of molar concentration of mRNA, and thus may serve as a site of regulation for the recruitment of mRNA into polysomes. Previous studies have indicated that phosphorylation of eIF-4E at Ser-53 is correlated with an increased rate of protein synthesis in a variety of systems in vivo and is required for eIF-4E to become bound to the 48S initiation complex. In this study we show that overexpression of eIF-4E in HeLa cells using an episomally replicating, BK virus-based vector leads to an unusual phenotype: cells grow rapidly, forming densely packed, multilayered foci. They progressively form syncytia, some containing as many as six nuclei, and ultimately lyse 1 month after transfection. Some of these properties are reminiscent of oncogenically transformed cells. Cells transfected with the identical vector expressing a variant of eIF-4E, which contains alanine at position 53 and thus cannot be phosphorylated at the major in vivo site, grow normally. Estimations using the Ala-53 variant or a bacterial chloramphenicol acetyltransferase reporter gene in the same vector indicate that the degree of eIF-4E overexpression is 3- to 9-fold more than the endogenous level. These results suggest that eIF-4E may play a key role in cell cycle progression.
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PMID:Overexpression of eukaryotic protein synthesis initiation factor 4E in HeLa cells results in aberrant growth and morphology. 212 55

The major late 16S mRNA species of simian virus 40 encodes both a 61-amino-acid protein, LP1, and the major virion protein, VP1. Although the initiation signal GCCAUGG is usually utilized at high efficiency, at least one-third of 40S ribosomal subunits bypass it when it is present on the major 16S mRNA of simian virus 40 (S. A. Sedman, P. J. Good, and J. E. Mertz, J. Virol. 63:3884-3893, 1989). The LP1 translation initiation codon is situated 10 bases from the 5' end of this mRNA. To determine whether the short length of the untranslated leader of this mRNA affects the efficiency of translation initiation at the LP1 initiation signal, monkey cells were transfected with plasmids which encode major late 16S-like mRNAs with 5' untranslated regions (UTRs) of 6 or 44 bases. Decreasing the length of the 5' UTR from 44 to 6 bases resulted in a 30% decrease in translation initiation at the LP1 AUG and a threefold increase in synthesis of VP1. When the VP1 open reading frame was replaced with the chloramphenicol acetyltransferase open reading frame, the reduction in 5' UTR length resulted in a 70% decrease in translation initiation at the LP1 AUG and a 30% increase in chloramphenicol acetyltransferase synthesis. Therefore, ribosomes bypass an AUG codon more efficiently when it is located very close to the 5' end of the mRNA.
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PMID:Translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' cap. 215 33

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.
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PMID:Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA. 284 Nov 27

The rpL34 gene, which encodes a cytoplasmic ribosomal protein with a high homology to the rat 60S r-protein L34, was isolated from a genomic library of tobacco (Nicotiana tabacum L. cv. Xanthi-nc). A 1500 bp upstream promoter fragment was fused to the chloramphenicol acetyltransferase (CAT) reporter gene or beta-glucuronidase (GUS) reporter gene and transferred into tobacco plants by the Agrobhacterium-mediated leaf disk transformation method. Analysis of CAT activity in leaf tissues showed that mechanical wounding increased the rpL34 promoter activity about 5 times as compared to untreated controls and that the promoter activity was further enhanced by plant growth regulators, 2,4-dichlorophenoxyacetic acid and benzyladenine. Histochemical GUS staining patterns of the transgenic plants showed that the rpL34 promoter activity is high in actively growing tissues, including various meristems, floral organs, and developing fruits. A series of 5' deletion analyses of the rpL34 promoter indicated that a 50 bp region located between -179 and -129 is essential for wound, auxin and cytokinin responses. Deletion of this region reduced the promoter activity to an undetectable level. Insertion of the 50 nucleotide sequence into a minimal promoter restored the promoter activity and the promoter strength was proportional to the copy number of the upstream sequence. The role of TATA and CAAT box regions was studied by a series of 3' deletion analyses. A 3' deletion up to -28 did not significantly affect the promoter strength. However deletion of the promoter up to 70 bp, which deleted the TATA box region, significantly reduced promoter activity. Further deletion of the promoter up to - 104. eliminating the CAAT box region, abolished the promoter activity. These results suggest that the TATA box and CAAT box regions are also important for the rpL34 promoter activity in addition to the 50 bp upstream region.
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PMID:Promoter elements controlling developmental and environmental regulation of a tobacco ribosomal protein gene L34. 900 4

Conditions are described for using a primer extension inhibition (toeprinting) assay to study the initiation step of protein synthesis in rabbit reticulocyte lysates. These studies revealed that chloramphenicol acetyltransferase mRNA, which is widely used as a reporter, forms unusually labile initiation complexes. This and other unexpected problems were solved by adjustments in pH and temperature during the reverse transcriptase step. Complications that may occur during the ribosome binding step were also examined, including the possibility of rapid mRNA degradation. The suitability of inhibitors commonly used to block the elongation phase of translation was studied. The refined toeprinting assay was used to confirm context-dependent selection of the AUG start codon. Absence of the m7G cap did not subvert the process wherein initiation is restricted to the first AUG codon. The fidelity of initiation was impaired, however, when NaF was introduced during the ribosome binding step. In a preliminary assessment of the processivity of scanning, no dissociation of 40S ribosomal subunits was detected as the distance from the cap to the AUG codon was increased to nearly 300 bases. With an mRNA that contains a pseudoknot upstream from the AUG codon, the toeprinting assay revealed 40S ribosomal subunits trapped behind the base paired structure. Thus the assay is usable for mapping some intermediates as well as for detecting conventional 80S initiation complexes.
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PMID:Primer extension analysis of eukaryotic ribosome-mRNA complexes. 977 44