Showing papers on "Small hairpin RNA published in 1996"

Role of the DIS hairpin in replication of human immunodeficiency virus type 1.

Abstract: The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the autocomplementary loop sequences. This hairpin motif with the palindromic loop sequence is referred to as the dimer initiation site (DIS), and the type of interaction is termed loop-loop kissing. Detailed phylogenetic analysis of the DIS motifs in different human and simian immunodeficiency viruses revealed conservation of the hairpin structure with a 6-mer palindrome in the loop, despite considerable sequence divergence. This finding supports the loop-loop kissing mechanism. To test this possibility, proviral genomes with mutations in the DIS palindrome were constructed. The appearance of infectious virus upon transfection into SupT1 T cells was delayed for the DIS mutants compared with that obtained by transfection of the wild-type provirus (pLAI), confirming that this RNA motif plays an important role in virus replication. Surprisingly, the RNA genome extracted from mutant virions was found to be fully dimeric and to have a normal thermal stability. These results indicate that the DIS motif is not essential for human immunodeficiency virus type 1 RNA dimerization and suggest that DIS base pairing does not contribute to the stability of the mature RNA dimer. Instead, we measured a reduction in the amount of viral RNA encapsidated in the mutant virions, suggesting a role of the DIS motif in RNA packaging. This result correlates with the idea that the processes of RNA dimerization and packaging are intrinsically linked, and we propose that DIS pairing is a prerequisite for RNA packaging.

A hairpin structure upstream of the terminator hairpin required for ribosomal protein L4-mediated attenuation control of the S10 operon of Escherichia coli.

Abstract: Ribosomal protein L4 of Escherichia coli regulates transcription of the 11-gene S1O operon by promoting premature termination of transcription (attenuation) at a specific site within the 172-base untranslated leader. We have analyzed the roles of various domains of the leader RNA in this transcription control. Our results indicate that the first 60 bases of the leader, forming the three proximal hairpin structures, are not essential for in vivo L4-mediated attenuation control. However, a deletion removing the fourth hairpin, which is immediately upstream of the terminator hairpin, eliminates L4's effect on transcription. Base changes disrupting complementarity in the 6-bp stem of this hairpin also abolish L4 control, but compensatory base changes that restore complementarity also restore L4's effect. In vitro transcription studies confirm that this hairpin structure is necessary for L4's role in stimulating transcription termination by RNA polymerase.

Improved Method for Selecting RNA-Binding Activities in vivo

Abstract: RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo. We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein.

Direct analysis of RNA after transfection.

Abstract: It is possible to transfect mammalian cells with a gene of interest and directly detect the RNA made from that gene 24 to 48 hr later. The ability to analyze the RNA directly allows mutation and functional analysis of an intact gene, as the promoter does not have to be subcloned. This technique is also essential when fusion genes are used. In order to be sure that the level of the reporter protein produced by a fusion gene provides an accurate measure of appropriately initiated RNA from the promoter under study, it is necessary to determine the amount and 5' end of the fusion message. An investigator who verifies this using direct RNA analysis can proceed with some confidence that the level of the reporter protein is a measure of promoter activity. The difficulty in directly analyzing RNA is that the sensitivity of detection is at the limits of present technology. Consequently, the transfection protocol, RNA preparation, and RNA analysis techniques all have to be working near optimum in order for an experiment to work. This overview discusses parameters that must be considered when directly analyzing RNA after a transfection: transfection efficiency, the method of preparation of the RNA, the method of analysis of RNA, and strength of the promoter.