Probing interactions between HIV-1 reverse transcriptase and its DNA substrate with backbone-modified nucleotides.
BACKGROUND: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extremely helpful. Often the question arises whether conformations obtained in this way appropriately reflect the reactivity of enzymes, however. Rates that characterize transitions are therefore compulsory experiments for the elucidation of the reaction mechanism. Such experiments have been performed for the reverse transcriptase of the type 1 human immunodeficiency virus (HIV-1 RT). RESULTS: We have developed a methodology to monitor the interplay between HIV-1 RT and its DNA substrate. To probe the protein-DNA interactions, the sugar backbone of one nucleotide was modified by a substituent that influenced the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide follows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar substituent and the enzyme. The model was confirmed by kinetic experiments with HIV-1 RT mutants. CONCLUSIONS: Experiments with HIV-1 RT demonstrate that strand-elongation efficiency using a modified nucleotide correlates well with distances between the DNA substrate and the enzyme. The functional group at the modified nucleotides acts as an 'antenna' for steric interactions that changes the optimal transition state. Kinetic experiments in combination with backbone-modified nucleotides can therefore be used to gain structural information about reverse transcriptases and DNA polymerases. (+info)
cAMP inhibits translation by inducing Ca2+/calmodulin-independent elongation factor 2 kinase activity in IPC-81 cells.
Treatment of IPC-81 cells led to inhibition of protein synthesis, which was accompanied by an increase in the average size of polysomes and a decreased rate of elongation, indicating that it involved inhibition of peptide chain elongation. This inhibition was also associated with increased phosphorylation of elongation factor eEF2 (which inhibits its activity) and enhanced Ca2+/calmodulin-independent activity of eEF2 kinase. Previous work has shown that phosphorylation of eEF2 kinase by cAMP-dependent protein kinase (cAPK) in vitro induces such activator-independent activity, and the present data show that such a mechanism can occur in intact cells to link physiological levels of cAPK activation with inhibition of protein synthesis. (+info)
Human immunodeficiency virus type 1 Tat-dependent activation of an arrested RNA polymerase II elongation complex.
The human immunodeficiency virus type 1 (HIV-1) Tat protein is a transcriptional activator that is essential for efficient viral gene expression and replication. Tat increases the level of full-length transcripts from the HIV-1 promoter by dramatically enhancing the elongation efficiency of the RNA polymerase II complexes assembled on this promoter. Tat could potentially activate the transcription machinery during initiation, elongation, or both. We used an immobilized HIV-1 promoter template with a reversible lac repressor (LacR) elongation block inserted downstream to dissect the stages in transcription affected by Tat. Transcription complexes assembled in the absence of Tat and blocked by LacR cannot be activated by incubation with Tat alone. These complexes can, however, be activated if Tat is added in combination with cellular factors. In this system, Tat also promoted the assembly of preinitiation complexes capable of elongating efficiently, suggesting that Tat can associate with transcription complex at an early stage. These data indicate that Tat can activate elongation of RNA polymerase by modifying an already elongating transcription complex. The data also suggest the possibility that Tat can interact with initiation complexes. (+info)
The semisynthesis of portions of hen's-egg lysozyme by fragment condensation.
1. We have evaluated several methods for coupling protected tryptic peptides and have selected one that is satisfactory in terms of efficiency and degree of racemization. 2. We report the use of the chosen method (which must be slightly modified for each application in the light of the result of micro-scale pilot experiments) for the preparation of five fragments of hen's-egg lysozyme ranging in length from 31 to 51 residues. 3. We report methods for the complete deprotection of these fragments. They have been characterized by end-group determination, amino acid analysis and tryptic digestion. (+info)
Regeneration of renal proximal tubules after mercuric chloride injury is accompanied by increased binding of aminoacyl-transfer ribonucleic acid.
Homogenates of rat kidney cortex obtained 1,3 or 14 days after a single injection of HgCl2 were used to prepare the post-microsomal pH5 supernatant fraction. The activity of this fraction for peptide synthesis from [14C]phenylalanyl-tRNA was significantly increased at 1 and 3 days, at which time the proximal tubules are regenerating [Cuppage & Tate (1967) Am. J. Pathol. 51, 405-429]. This increased activity could not be attributed to a decreased inhibitory activity, but was due to an increased aminoacyl-tRNA binding, i.e. elongation-factor-1 activity, in the supernatant fraction. (+info)
The effect of template RNA structure on elongation by HIV-1 reverse transcriptase.
Reverse transcription of the RNA genome of retroviruses has to proceed through some highly structured regions of the template. The RNA genome of the human immunodeficiency virus type 1 (HIV-1) contains two hairpin structures within the repeat (R) region at the 5' end of the viral RNA (Fig. 1Fig. 1Template RNA structure of the HIV-1 R region and the position of reverse transcription pause sites. The HIV-1 R region (nucleotides +1/97) encodes two stable RNA structures, the TAR and polyA hairpins . The latter hairpin contains the AAUAAA hexamer motif (marked by a box) that is involved in polyadenylation. The lower panel shows the predicted structures of the wild-type and two mutant forms of the polyA hairpin that were used in this study. Nucleotide substitutions are boxed, deletions are indicated by black triangle. The thermodynamic stability (free energy or DeltaG, in kcal/mol) was calculated according to the Zucker algorithm . The TAR hairpin has a DeltaG of -24.8 kcal/mol. Minus-strand DNA synthesis on these templates was initiated by a DNA primer annealed to the downstream PBS. The position of reverse transcription pause sites observed in this study are summarized. All numbers refer to nucleotide positions on the wild-type HIV-1 transcript. Filled arrows represent stops observed on the wild-type template, and open arrows mark the pause sites that are specific for the structured A-mutant template. The sizes of the arrows correspond to the relative frequency of pausing. Little pausing was observed on the B-mutant template with the destabilized polyA hairpin.). These structures, the TAR and polyA hairpins, fulfil important functions in the viral life cycle. We analyzed the in vitro elongation properties of the HIV-1 reverse transcriptase (RT) enzyme on the wild-type RNA template and mutants thereof with either a stabilized or a destabilized polyA hairpin. Stable RNA structure was found to interfere with efficient elongation of the RT enzyme, as judged by the appearance of pause cDNA products. A direct relation was measured between the stability of template RNA structure and the extent of RT pausing. However, the position of structure-induced pause sites is rather diverse, with significant stops at a position approximately 6 nt ahead of the basepaired stem of the TAR and polyA hairpins. This suggests that the RT enzyme is stalled when its most forward domain contacts the RNA duplex. Addition of the viral nucleocapsid protein (NC) to the in vitro assay was found to overcome such structure-induced RT stops. These results indicate that the RT polymerase has problems penetrating regions of the template with stable RNA structure. This effect was more pronounced at high Mg2+ concentrations, which is known to stabilize RNA secondary structure. Such a structure-induced defect was not apparent in reverse transcription assays performed in virus-infected cells, which is either caused by the NC protein or other components of the virion particle. Thus, retroviruses can use relatively stable RNA structures to control different steps in the viral life cycle without interfering with the process of reverse transcription. (+info)
A new concept of the function of elongation factor 1 in peptid chain elongation.
An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments in which mixtures of [3H]EF-1, ribosomes from Drebs II ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound. (+info)
Sensitized photomodification of mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases.
To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A. (+info)