Biomedical subjects

D J Goss

Publications and source records attributed to D J Goss.

At least 37 records · Page 2Linked to original sources

PubMed1993

Strategies for the computation of infrared CD and absorption spectra of biological molecules: ribonucleic acids.

We report observed and computed infrared (vibrational) circular dichroism spectra of a number of polyribonucleic acids in aqueous solutions in the 1600-1750 cm-1 spectral region, in which C = O and some nucleotide base ring stretching vibrations occur. The experimental data are compared with results calculated using different levels of sophistication within the exciton approach. We find that observed band shapes are generally well reproduced by these models, particularly if care is taken to determine the direction of the vibrational dipole transition moments accurately.

Biophysical Phenomena↗

PubMed1992

Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.

The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.

Animals↗

PubMed1992

Characterization of the interaction of wheat germ protein synthesis initiation factor eIF-3 with mRNA oligonucleotide and cap analogues.

Direct fluorescence titration experiments of wheat germ protein synthesis initiation factor eIF-3 with mRNA cap and oligoribonucleotide analogues were performed in order to determine the equilibrium association constants (Keq) for the eIF-3.mRNA interaction as a function of pH and temperature. These data suggest that (i) the eIF-3.mRNA interaction is not cap-specific (i.e., m7G-specific), (ii) ATP hydrolysis is not involved in the interaction, and (iii) the interaction is primarily ionic in nature. Competition experiments between a rabbit alpha-globin mRNA oligoribonucleotide analogue and either mRNA cap analogues or nucleoside triphosphates (NTPs) are also reported; these experiments indicate that NTPs act as both activators and competitive inhibitors of the mRNA.eIF-3 association. The results are consistent with a partially uncompetitive binding mechanism, whereby at low NTP concentrations (less than or equal to 10 microM) the bound NTP enhances subsequent mRNA binding to eIF-3, perhaps by inducing a conformational change, and at higher NTP concentrations, the NTP acts as a competitive inhibitor for the mRNA binding site on eIF-3.

Adenosine Triphosphate↗

PubMed1991

Interaction of wheat germ protein synthesis initiation factors eIF-3, eIF-(iso)4F, and eIF-4F with mRNA analogues.

The interaction of wheat germ eIF-3 with the wheat germ cap-binding proteins eIF-(iso)4F and eIF-4F as a function of pH and ionic strength is described. Direct fluorescence titration experiments are used to measure the equilibrium association constants (Keq) for the binary protein/protein complexes as well as for the interaction of eIF-3 with methylated cap analogues and rabbit alpha-globin mRNA oligonucleotide analogues. The Keq values for ternary eIF-3/eIF-(iso)4F/analogue and eIF-3/eIF-4F/analogue interactions were also measured. The equilibrium binding constants were used to calculate coupling free energies, which provide an estimate of the cooperativity for the interaction of the mRNA analogues, eIF-3, and either eIF-4F or eIF-(iso)4F. These data suggest a mechanism in which the binding of eIF-(iso)4F or eIF-4F to mRNA enhances the subsequent binding of eIF-3 to the message. This may lead to favorable positioning of the complex on the ribosome and thereby enhance translation.

Animals↗

PubMed1991

Wheat germ initiation factors 4F and (iso)4F interact differently with oligoribonucleotide analogues of rabbit alpha-globin mRNA.

The binding of capped oligoribonucleotide analogues of the 5' terminus of rabbit alpha-globin mRNA to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F was measured by direct fluorescence techniques. An analysis of the equilibrium association constants (Keq) indicates that both eIF-4F and eIF-(iso)4F recognize primarily the m7G cap structure but differ in the recognition of other structural features. eIF-4F is sensitive to the position and sequence of hairpin structures within the oligoribonucleotide, while eIF-(iso)4F shows a preference for linear sequences. These differences suggest that wheat germ eIF-4F and eIF-(iso)4F may have discriminatory activity for mRNA recognition.

Animals↗

PubMed1991

A comparison of the binding of methylated cap analogues to wheat germ protein synthesis initiation factors 4F and (iso)4F.

The binding of the 5'-terminal cap analogues m7GpppG and m7GTP to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F as a function of pH, ionic strength, and temperature is described. Equilibrium binding data indicate that eIF-4F and eIF-(iso)4F have different mechanisms for interacting with the 5'-cap structure, but the complexes formed between m7GpppG and wheat germ factor eIF-(iso)4F more closely resemble complexes formed between this cap analogue and either mammalian eIF-4E or eIF-4F. The binding of these initiation factors to the hypermethylated cap analogues m2,7GMP, m2,7GpppG, and m2,2,7GpppG is also investigated. The differences in affinity of eIF-4F and eIF-(iso)4F for the hypermethylated 5'-terminal cap structures suggest that these factors may have discriminatory activity.

Eukaryotic Initiation Factor-4F↗

PubMed1990

A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs.

The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.

Animals↗

PubMed1990

DNA solution conformation via infrared circular dichroism: experimental and theoretical results for B-family polymers.

Infrared (vibrational) circular dichroism (VCD) has been observed for the DNA models d(CG)5, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) in the B-conformation in buffered, aqueous solution. The observed results are quantitatively interpreted in terms of the exciton model for coupled carbonyl stretching vibrational states.

Circular Dichroism↗

PubMed1990

Fluorescence study of the binding of m7GpppG and rabbit globin mRNA to protein synthesis initiation factors 4A, 4E, and 4F.

The interactions of protein synthesis initiation factors eIF-4E from human erythrocytes and eIF-4A and eIF-4F from rabbit reticulocytes with the cap analogue m7GpppG and rabbit globin mRNA were investigated. The equilibrium binding constants for the binary complex formation of eIF-4E-eIF-4A, m7GpppG-eIF-4E, m7GpppG-eIF-4F, globin mRNA-eIF-4E, globin mRNA-eIF-4F, and globin mRNA-eIF-4A were measured by direct fluorescence titration experiments. The binding of eIF-4E to globin mRNA was found to be 5.5-fold tighter than its binding to m7GpppG; the binding of eIF-4F for globin mRNA and m7GpppG was similar to that of eIF-4E. Association equilibrium constants were determined for the ternary system mRNA-eIF-4E-eIF-4A; four thermodynamically independent equilibria characterize the system. These equilibrium binding constants were used to calculate coupling free energies, which provided an estimate of the cooperativity of the interaction of eIF-4E, eIF-4A, and mRNA. These coupling energies were all found to be small and positive, indicative of anticooperative binding.

Binding Sites↗

PubMed1990

A spectroscopic study of the binding of N-7-substituted cap analogues to human protein synthesis initiation factor 4E.

The binding of N-7-substituted cap analogues to eIF-4E from human erythrocytes is described. Data presented here indicate that there is a correlation between the tightness of binding of these cap analogues to eIF-4E and their potency as inhibitors of protein synthesis. This result indicates that the inhibitory activity of the cap analogues is strictly a function of the affinity of the analogue for eIF-4E under equilibrium conditions. The pH dependence of binding of the cap analogues to eIF-4E indicates that the enolate form of the cap is preferred, as originally postulated by Rhoads et al. [(1983) Biochemistry 22, 6084-6088]. Data indicate that there are differences in the mode of binding of alkyl-substituted and aryl-substituted cap analogues to eIF-4E arising from favorable interactions of the phenyl ring with the guanosine moiety. These differences may explain the enhanced recognition of the aryl-substituted cap analogues by eIF-4E.

Cross-Linking Reagents↗

PubMed1989

A spectroscopic study of the binding of m7GTP and m7GpppG to human protein synthesis initiation factor 4E.

The binding of analogues of the 7-methylguanosine-containing cap, m7GTP and m7GpppG, to eIF-4E from human erythrocytes as a function of pH, temperature, and ionic strength is described. From the pH-dependent binding of m7GTP and m7GpppG to eIF-4E, a new model describing the nature of the cap.eIF-4E interaction is proposed. The thermodynamic values and ionic strength dependence of binding are consistent with a binding site which is primarily hydrophobic. Fluorescence and circular dichroism data indicate that tryptophan residues may be involved in base-stacking interactions with the cap in a somewhat buried environment. The model presented here confirms the earlier proposal [Rhoads et al. (1983) Biochemistry 22, 6084-6088] that the enolate tautomer of the cap is preferred for interaction and further proposes that the interaction is with a protonated amino acid residue, such as histidine, while stacking with an aromatic amino acid, such as tryptophan.

Circular Dichroism↗

PubMed1989

IR vibrational CD in model deoxyoligonucleotides: observation of the B----Z phase transition and extended coupled oscillator intensity calculations.

The first observation of ir vibrational CD (VCD) in small model DNA molecules is reported. The VCD signals in the 1550-1750-cm-1 spectral region, which originate from coupling of carbonyl stretching modes of the nucleic acid bases, are found to be sensitive to the handedness of the polymer helix. The formalism to calculate VCD intensities of polymers is developed from the exciton model derived earlier by Tinoco [(1963) Radiation Res. 20, 133; (1960) J. Chem. Phys. 33, 1332; (1964) J. Am. Chem. Soc. 86, 297] and Schellman and co-workers [(1975) Biopolymers 14, 173; (1969) J. Phys. Chem. 73, 28]. The resulting equations, which are a direct extension of the dimeric case known as the "coupled oscillator," are used in model calculations of the helical polymers.

Circular Dichroism↗

PubMed1989

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.

Escherichia coli↗

PubMed1988

A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes.

The rate constants for eucaryotic initiation factor 3 (eIF3) association and dissociation with 40S ribosomal subunits and 80S monosomes have been determined. These rate constants were determined by laser light scattering with unmodified eIF3. The affinity of eIF3 for 40S subunits is about 30-fold greater than for 80S ribosomes. This difference in affinity resides mainly in the association rate constants. Rate constants of 8.8 X 10(7) and 7.3 X 10(6) M-1 s-1 were obtained for eIF3 binding to 40S subunits and 80S ribosomes, respectively. From thermodynamic cycles, the affinity of eIF3-40S subunits for 60S subunits is about 30-fold lower than free 40S subunits for 60S subunits. A calculation shows that under these conditions and assuming simple equilibria, approximately 12% of ribosomal subunits would associate via a reaction of 40S-eIF3 with 60S subunits as opposed to a path where eIF3 dissociates from the 40S subunits prior to association with 60S subunits.

Eukaryotic Initiation Factor-3↗

PubMed1988

Effects of eucaryotic initiation factor 3 on eucaryotic ribosomal subunit equilibrium and kinetics.

In order to understand the possible role of eucaryotic initiator factor 3 (eIF-3) in maintaining a pool of eucaryotic subunits, we have measured the effects of eIF-3 on the equilibria and kinetics of ribosomal subunit association and dissociation. The ribosomal subunit interactions have been studied by laser light scattering, which does not perturb the system. We find that eIF-3 reduces the apparent association rate of reticulocyte, wheat germ, and Artemia ribosomes. The kinetics of the reassociation for a shift in [Mg2+] from 0.5 to 6 mM are best explained by a model where eIF-3 dissociates from the 40S subunits prior to association of the 40S and 60S subunits. Static titrations indicate there is some binding of eIF-3 to 80S ribosomes at lower [Mg2+].

Animals↗

PubMed1987

A fluorescence study of the binding of eucaryotic initiation factors to messenger RNA and messenger RNA analogues.

The binding of the eucaryotic polypeptide chain initiation factors (eIFs) 4A, 4B, and 4F to poly(1,N6-ethenoadenylic acid) [poly(epsilon A)] was investigated by fluorescence spectroscopy. Competition experiments allowed us to determine the relative affinity of these proteins for mRNA cap analogues and the triplets AUG, GUG, UUU, UAA, and UGA. The salt dependence of eIF-4A binding to poly(epsilon A) and mRNA suggested that the binding was largely electrostatic and was enhanced in the presence of Mg2+ and ATP. The size of the binding site of eIF-4A, eIF-4B, and eIF-4F on poly(epsilon A) was approximately 13, 25, and 35 nucleotides, respectively. Fluorescence studies with the cap analogue 7-methylguanosine triphosphate as well as competition studies with poly(epsilon A) provide further evidence for a direct interaction of eIF-4F with the cap region. There was no evidence that either eIF-4B or eIF-4A bound the mRNA cap directly. In contrast to the other two factors, eIF-4B was found to bind preferentially to AUG, and of all the triplets tested, AUG was the most effective competitor for poly(epsilon A) binding.

Eukaryotic Initiation Factor-4A↗

PubMed1986

Magnesium ion dependent equilibria, kinetics, and thermodynamic parameters of Artemia ribosome dissociation and subunit association.

The influence of magnesium ion concentration on the equilibrium and kinetics of Artemia ribosome dissociation and subunit association has been studied by laser light scattering. Ribosomal aggregation was found to be reduced by addition of 0.1-0.05 mM spermidine and KCl concentrations of 100 mM. The ribosomes were found to be stable at low [Mg2+], and the curves obtained for ribosome-subunit equilibrium were independent of the direction and origin of the magnesium ion titration. Thermodynamic parameters were obtained from the temperature-dependent equilibria and have been compared to those of wheat germ and Escherichia coli type A ribosomes. The entropy term calculated for the association of 40S and 60S subunits is small, and the reaction is exothermic. The entropy term is negative, favoring subunit dissociation, and contributes less to the free energy than the enthalpy term. Rate constants for ribosome dissociation and subunit association have been determined. The reaction curves gave no evidence for sequential processes and were homogeneous.

Animals↗

PubMed1984

Cooperative interactions in the system ribosomes-ribosomal protein S1-polynucleotide triplets.

Association equilibria have been determined in the ternary system uridyl triplets (T)-ribosomal protein S1 (S)-ribosomes (Rb) depleted of S1 at 6 and 10 mM Mg2+. For 1:1 stoichiometry of reactants, four thermodynamically independent equilibria characterize the ternary system. The binary interaction Rb + T was studied by following the fluorescence quenching of labeled ribosomes by added T. The Rb + T association constant for UpUpUp triplets was 10-20-fold greater than for ApUpG triplets. The interaction Rb + S was studied by following the changes in fluorescence anisotropy when labeled S1 reacted with ribosomes. The remaining two independent equilibrium constants (for S + T and RbT + S) were obtained from fits to observed anisotropy measurements when varying amounts of T were added to a solution of ribosomes and fluorescently labeled S1. This indirect procedure allows one to measure S + T binding, an association that is difficult to determine directly. Over the concentration interval 5-10 mM Mg2+, the association constant for Rb + S increases with the sixth power of [Mg2+], whereas the association constant for S + T decreases approximately 2-fold as Mg2+ is increased from 6 to 10 mM Mg2+. T binds to Rb more tightly at 10 mM than at 6 mM Mg2+. When S1 is bound to Rb, however, at 10 mM Mg2+ the binding constant for T is decreased 10-fold and the Mg2+ dependence is reversed. These interactions can be described in terms of coupling free energies. For the ternary complex, three linearly independent coupling free energies can be written.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗