Biomedical subjects

E Skripkin

Publications and source records attributed to E Skripkin.

24 records · Page 2Linked to original sources

PubMed1994

Mutational analysis of the bipartite dimer linkage structure of human immunodeficiency virus type 1 genomic RNA.

The genome of all retroviruses consists in two homologous RNA molecules associated near their 5' end in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for several functions of the retroviral cycle such as encapsidation, reverse transcription, and translation. In human immunodeficiency virus type 1 (HIV-1), a region downstream of the splice donor site was initially postulated to mediate dimerization. However, we recently showed that the dimerization initiation site is located upstream of the splice donor site and suggested that dimerization may initiate through a loop-loop interaction. Here, we show that single base mutations in the palindromic loop of the dimerization initiation site completely abolish dimerization, while introduction of compensatory mutations restores the process. Furthermore, two single nucleotide mutants that are unable to form homodimers efficiently codimerize, while the wild type RNA and the compensatory mutant, which both form homodimers, are unable to codimerize. These results unambiguously prove the interaction between the palindromic loops of each monomer. By contrast, none of the deletions that we introduced downstream of the splice donor site abolishes dimerization. However, deletions of two purine tracts located in the vicinity of the initiation codon of the gag gene significantly decrease the thermal stability of the HIV-1 RNA dimer.

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PubMed1994

Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides 1-707 of HIV-1 RNA. These nucleotides map exclusively in a restricted area upstream of the splice donor site and downstream of the primer binding site. They are centered around a palindromic sequence (GUGCAC279) located in a hairpin loop. Our results support a model in which dimer formation is initiated by the annealing of the palindromic sequences, possibly by a loop-loop interaction between the two monomers. Further experiments show that the deletion of the stem-loop or base substitutions in the loop abolish dimerization, despite the presence of the previously postulated dimer linkage structure. On the other hand, deletions of the purine tracts downstream of the splice donor site do not prevent dimerization. Therefore, we conclude that the palindromic region represents the dimerization initiation site of genomic RNA.

Base Sequence↗

PubMed1994

Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site.

The retroviral genome consists of two homologous RNA molecules associated close to their 5' ends. We studied the spontaneous dimerization of four HIV-1 RNA fragments (RNAs 1-707, 1-615, 311-612, and 311-415) containing the previously defined dimerization domain, and a RNA fragment (RNA 1-311) corresponding to the upstream sequences. Significant dimerization of all RNAs is observed on agarose gels when magnesium is included in the electrophoresis buffer. In contrast to dimerization of RNAs 311-612 and 311-415, dimerization of RNAs 1-707, 1-615 and 1-311 strongly depends on the size of the monovalent cation present in the incubation buffer. Also, dimerization of RNAs 1-707, 1-615, and 1-311 is 10 times faster than that of RNAs 311-612 and 311-415. The dimers formed by the latter RNAs are substantially more stable than that of RNA 1-615, while RNA 1-311 dimer is 5-7 degrees C less stable than RNA 1-615 dimer. These results indicate that dimerization of HIV-1 genomic RNA involves elements located upstream of the splice donor site (position 305), i.e. outside of the previously defined dimerization domain.

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PubMed1990

Transition of the mRNA sequence downstream from the initiation codon into a single-stranded conformation is strongly promoted by binding of the initiator tRNA.

Using an RNA footprinting technique, accessible sites on the mRNA initiation region bound to the ribosome have been determined. Chemical probing experiments have been done both in the presence and absence of the initiator tRNA with dimethyl sulfate, kethoxal and carbodiimide as reagent probes. As an mRNA, a mini-mRNA containing the initiation region of bacteriophage lambda gene cro has been used. This region is characterized by a long single-stranded Shine-Dalgarno (SD) sequence followed by two hairpin structures of which the first one comprises in its loop the initiation codon. As compared to a free mRNA, the only nucleotides additionally protected in the binary mRNA-ribosome complex have been those which belong to the S-D sequence and the initiation codon. The protection of other nucleotides has not changed. Addition of the initiator RNA results in the modification of nucleotides in the stems of the downstream hairpin structures of the initiation region. This reflects their transition into a single-stranded conformation promoted by tRNA. A possible implication of these findings for the decoding process is discussed.

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PubMedDate not supplied

The stress-related production of the active Photinus pyralis and Luciola mingrelica firefly luciferases in Escherichia coli.

The kinetics of Photinus pyralis and Luciola mingrelica luciferase gene expression was studied on plasmids with the thermoinducible lambda PR promoter in Escherichia coli by SDS-gel electrophoresis of cell lysates to follow luciferase protein-synthesized, enzyme immunoassay (EIA) to follow native enzyme conformer, and the luciferase activity assay. E. coli cells were cultivated at temperature schemes 28-42-21 degrees C or 28-21 degrees C, or at alkali pH shift. In the cases of thermoinduction and pH shift, the luciferase expressions have similar features. The 3-h thermoinduction (42 degrees C) followed by the incubation at 21 degrees C, for 10 h resulted in the maximal amount of the luciferase protein of 4-5% of the total cell proteins. The yield did not change further. The amount of native luciferase conformer and the luciferase activity started to grow after incubation for 10 h at 21 degrees C and reached the maximum after 50-60 h when the synthesized luciferase protein adopted the native-like conformation. At the same time, only 50% of the latter appeared to be catalytically active. An increase in the enzymatic activity correlates with an increase in the intracellular pH and ATP content. Intracellular metabolic reactions were shown to play a role in the conformational changes of the enzyme in a postthermoinduction period, and a possible mechanism of this effect is proposed.

Animals↗

PubMedDate not supplied

Synthesis and ribosome binding properties of model mRNAs modified with undecagold cluster.

The synthesis and purification of short model messenger RNAs modified with undecagold cluster are described. A monoamino undecagold cluster was introduced on the oxidized 3' cis-glycol group of the mRNA followed by reduction of the formed Schiff's base. The stability of the modified mRNA under the conditions used for in vitro messenger RNA translation is studied. The possibility of the formation of a specific translational initiation complex with bacterial ribosomes and modified mRNAs is shown. The results of these experiments indicate that the attachment of an undecagold cluster to a mRNA is a useful tool for electron microscopic and crystallographic studies.

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