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Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 217 records · Page 12Linked to original sources
PubMed1989

Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF-4E, from the yeast Saccharomyces cerevisiae.

CD studies have shown that five tryptophan to phenylalanine (W----F) mutants of eukaryotic initiation factor-4E (eIF-4E) contain low amounts of alpha-helix, the main elements of secondary structure being beta-sheets/turns and aperiodic regions. Interactions with the cap analog m7GpppG are accompanied by changes in overall secondary structure which include reductions, and in one case an increase in alpha-helix content, as well as increases in total beta-structure (3 mutant forms) and decreases in total beta-structure (2 mutant forms). These changes may also involve more significant perturbations of localized regions containing phenylalanine residues either involved in nucleotide binding, or close to the nucleotide-binding site. Measurements of intrinsic Trp fluorescence have shown different quantum yields and reduced m7GpppG-induced quenching (with one exception). Acrylamide quenching studies yielded similar parameters for 4 of the mutants but 1 form displayed significantly reduced values. Melting experiments showed that the Trp fluorescence of 4 of the mutants decreased as the temperature was increased, this effect being reduced in 3 cases in the presence of m7GpppG. W 58 F showed an increase in fluorescence as the temperature was raised and this effect was accentuated in the presence of nucleotide. A preliminary attempt has been made to correlate the spectroscopic data with the known biological importance of the individual Trp residues.

Acrylamides↗
PubMed1989

Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: a novel translational control mechanism.

All mRNAs of human immunodeficiency virus 1 (HIV-1) contain in their 5' untranslated region a sequence termed TAR that responds to trans-activation by the tat (trans-activating) protein. This RNA sequence assumes a stable secondary structure, and its cap structure is relatively inaccessible. Here we report that these structural properties of the TAR sequence underlie the ability of TAR to inhibit in trans the translation of other mRNAs. This mechanism of translation inhibition involves the activation of the double-stranded RNA-dependent kinase (dsl), which in turn phosphorylates the protein synthesis initiation factor 2 (eIF-2). Mutations in the TAR region that diminish the stability of the secondary structure cause a significant reduction in the trans-inhibition. A similar reduction in the dsl activation occurs when TAR is placed further downstream of the cap structure. This is a clear demonstration of a specific naturally occurring mRNA sequence that can activate dsl. We suggest a novel translational regulatory mechanism that interdigitates the activities of eIF-2 and eIF-4F.

Animals↗
PubMed1989

Poliovirus translation: a paradigm for a novel initiation mechanism.

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.

Humans↗
PubMed1989

Construction of a fused LuxAB gene by site-directed mutagenesis.

Bacterial luciferases are heteropolymetric enzymes consisting of two non-identical subunits (alpha and beta). The two polypeptides are produced by transcription in the same direction of two genes, luxA and luxB, located immediately adjacent to each other and separated by only 29 base pairs in the Vibrio harveyi genome. Using site-directed mutagenesis, stop codons after luxA were eliminated and the luxB gene was placed in-frame with luxA, resulting in a fused luxAB gene. Transcription of two luxAB mutant genes from the bacteriophage T7 promoter and translation in Escherichia coli resulted in the synthesis of fused polypeptides containing the alpha and the beta subunits of luciferase linked by either a single amino acid residue or a decapeptide. E. coli synthesizing the latter fusion protein with the decapeptide linker expressed a level of luminescence comparable to E. coli containing the wild type genes while E. coli synthesizing the polypeptide with a single amino acid as a linker expressed about 2000-fold lower light. These results provide the basis for generating a bacterial luciferase system that can be expressed under the control of a single promoter in both eukaryotic and prokaryotic systems.

Base Sequence↗
PubMed1989

A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation.

Initiation of translation on poliovirus mRNA occurs by internal binding of ribosomes to a region within the 5'-noncoding portion of the mRNA. The mechanistic details and trans-acting factors involved in this event are not understood fully. We used a mobility-shift electrophoresis assay to identify a specific RNA-protein complex, which can form between an RNA fragment that contains nucleotides 559-624 of the poliovirus 5' UTR (untranslated region) and a component or components of a HeLa cell extract. Complex formation was reduced greatly in a reticulocyte lysate or a wheat-germ extract. A 52-kD polypeptide (p52) has been identified as part of the protein-RNA complex by use of an UV cross-linking assay. This polypeptide apparently is not a known translation initiation or elongation factor. The possible involvement of p52 in translation initiation of poliovirus protein synthesis is discussed.

Base Sequence↗
PubMed1989

Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts.

Translation initiation on poliovirus mRNA in poliovirus-infected cells has been shown to occur by internal binding of ribosomes to the 5' noncoding region (J. Pelletier and N. Sonenberg, Nature [London] 334:320-325, 1988). Here we show that internal ribosome binding can occur in HeLa cell extracts in vitro. Internal binding to the 5' noncoding region of poliovirus mRNA in a bicistronic context was independent of the upstream open reading frame and did not require poliovirus proteins.

HeLa Cells↗
PubMed1989

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Amino Acid Sequence↗
PubMed1989

A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity.

Eucaryotic initiation factor 4A (eIF-4A) is a member of a family of proteins believed to be involved in the ATP-dependent melting of RNA secondary structure. These proteins contain a derivative of the consensus ATP-binding site AXXGXGKT. To assess the importance of the consensus amino acid sequence in eIF-4A for ATP binding, we mutated the consensus amino-proximal glycine and lysine to isoleucine and asparagine, respectively. The effect of the mutations was examined by UV-induced cross-linking of [alpha-32P]dATP to eIF-4A. Mutation of the lysine residue (but not of the glycine residue) resulted in the loss of [alpha-32P]dATP cross-linking to eIF-4A, suggesting that the lysine is an important determinant in ATP binding to eIF-4A.

Adenosine Triphosphate↗
PubMed1989

Identification of a protein that binds specifically to RNA from the first exon of c-myc.

A 55 kilodalton protein present in HeLa and MEL cells, which binds specifically to RNA derived from the first exon of the human c-myc gene, has been identified using a UV-induced crosslinking assay. This protein, called p55, is found in both cytoplasmic and nuclear fractions. The binding site on the RNA, defined using deletion analysis and synthetic oligoribonucleotides, is a purine rich region located between the two major transcriptional initiation sites P1 and P2. Possible involvement of p55 in a number of regulatory events is discussed.

Animals↗
PubMed1988

High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin.

Numerous studies have established the important role that eukaryotic initiation factor-4E (eIF-4E) plays during protein biosynthesis. However, biochemical characterization of eIF-4E has proved difficult, mainly because of its low abundance in cells. To facilitate studies on eIF-4E, we have overexpressed Saccharomyces cerevisiae eIF-4E in Escherichia coli. The isolation of eIF-4E was simplified by using a cap-analog affinity matrix (agarose resin) that is considerably less demanding to prepare than those previously reported. We describe a simple and rapid purification scheme that can yield 2-5 micrograms of a homogenous and active preparation of eIF-4E from 1 ml of E. coli culture. E. coli-expressed eIF-4E is active as determined by its ability to bind the cap structure. The results demonstrate that the cap-binding activity of eIF-4E is not dependent on the presence of other proteins that are present at low levels in eIF-4E preparations isolated from eukaryotic cells.

Base Sequence↗
PubMed1988

Site-directed mutagenesis of the tryptophan residues in yeast eukaryotic initiation factor 4E. Effects on cap binding activity.

Initiation factor 4E is a 24-kilodalton polypeptide that binds specifically to the 5' cap structure of eukaryotic mRNAs. Sequence analysis of cDNA clones of initiation factor 4E from several species revealed a high tryptophan content (8 residues). Strikingly, all tryptophans are conserved evolutionarily in number and position between yeast and mammals. Here we show, using site-directed mutagenesis, that two of the tryptophans (those referred to as numbers 1 and 8) are absolutely required for the cap binding activity of an Escherichia coli expressed initiation factor 4E.

Amino Acid Sequence↗
PubMed1988

Circular dichroism and fluorescence studies on protein synthesis initiation factor eIF-4E and two mutant forms from the yeast Saccharomyces cerevisiae.

Circular dichroism studies have shown that eukaryotic initiation factor 4E contains low amounts of alpha-helix; the main elements of secondary structure are beta-sheets/turns and aperiodic regions. Interactions with cap analogs are accompanied by small but reproducible changes in overall secondary structure, which may also involve more significant perturbations of localized regions containing certain phenylalanine residues. Dissociation constants for interactions with nucleotides have been established from fluorescence titrations. Results show that the (N-7) methylated guanosine nucleotides bound more strongly than their nonmethylated counterparts. Involvement of a key tryptophan residue in the cap binding site was suggested. Additional studies with two cap binding mutant forms of the protein, designated SK-4 (W----75----L) and SK-6 (W----115----L), confirmed and extended these observations. Fluorescence melting experiments indicated that binding of cap analogs stabilized the protein against thermal perturbation and demonstrated subtle differences in folding between the wild-type and mutant forms of the protein. These subtle differences in folding may account for the observed loss in cap specificity of both mutant forms.

Amino Acid Sequence↗
PubMed1988

Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region.

The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirus allows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to a reporter gene and transfected into cells. There was extensive augmentation of the expression of this gene by poliovirus-mediated inhibition of cap-dependent protein synthesis. That the construct initiated in a cap-independent manner was verified through in vitro experiments. Small lesions throughout region P blocked its initiation function, implying that a coherent functional unit, hundreds of nucleotides long, is responsible for cap-independent initiation by poliovirus RNA.

Acetyltransferases↗
PubMed1988

Higher order structures of the 5'-proximal region decrease the efficiency of translation of the porcine pro-opiomelanocortin mRNA.

The SP6 polymerase/promoter system was used to synthesize porcine pro-opiomelanocortin mRNAs with nucleotide sequence deletions in the 5'- as well as 3'-untranslated and coding regions. The translational efficiency of the mutant mRNAs was evaluated by cell-free translation or by monitoring the rate and extent of ribosome binding in the presence of sparsomycin. The results of these experiments indicate that specific nucleotide sequences in the 5'-untranslated and coding regions of the pro-opiomelanocortin mRNA decrease its rate of translation. Structure mapping of the mRNA with double-strand and single-strand specific nucleases suggests that these sequences can form stable secondary structures.

Animals↗
PubMed1988

Mutational analysis of the 5' non-coding region of human immunodeficiency virus type 1: effects of secondary structure on translation.

The first 111 nt from the 5' end of human immunodeficiency virus type 1 (HIV-1) mRNAs are shown to have a strong inhibitory effect on the translation of mRNA in in vitro translation extracts as well as in Xenopus oocytes. Mutations in the sequence of the 5' untranslated region (UTR) designed to disrupt predicted secondary structure of this region relieve the inhibition. Inhibition is restored by mutations that reconstruct the predicted secondary structure. The accessibility of the 5'-terminal cap structure was also found to be increased by some of these mutations. We conclude that secondary structure in the 5' UTR of HIV-1 mRNAs and resultant inaccessibility of the cap structure is responsible for the inhibition of translation. The implications of these findings for the understanding of the life cycle of HIV-1 are discussed.

Animals↗