This laboratory will investigate the transcriptional control mechanisms of ribosomal RNA (rrn) operons of E. coli. We will employ recently developed genetic methods and assays capable of assaying expression of individual rrn operons. We will attempt to determine how rrn operons can be transcribed without coupling to translation. These studies are a continuation of our previous work which showed that Tn9, TN10, and IS1 are not polar in rrn operons, although these DNA elements are polar in operons that code for proteins. Read-through of Tn9, TN10, and IS1 will be confirmed by analysis of fusion transcripts and protein overproduction from within the transposable elements. The mechanism that allows read-through will be clarified by use of operon fusions that consist of ara or rrn promotors fused to lac or rrn sequences with and without inserts of Tn9, Tn10, and IS1, followed by polarity analysis. Another project will be in vitro mutagenesis to inactive one of the tandem rrn promotors, to allow subsequent classical genetic analysis of the rrn promotor region and functional analysis of the individual promotors. Our ability to construct mutations in vitro has been aided by our previous construction of a series of plasmids with internal deletions within rrn operons. These plasmids will be used to create an imbalance of rRNA synthesis, so that we can examine the resulting coordination or lack of coordination of ribosomal protein synthesis, thereby testing the hypothesis that ribosomal proteins translationally regulate their own mRNAs. These experiments could also discover that rRNA abundance regulates ribosomal protein genes that are not yet known to be translationally regulated. We will also attempt to isolate mutations in ribosomal RNA genes that confer resistance to antibiotics. This will allow establishment of adequate genetics in rrn operons. We have recently isolated the first antibiotic resistance mutation in any bacterial operon by use of a conceptually new selection that should allow isolation of additional mutations. We will determine the site of the mutations by genetic methods and DNA sequencing.
| Pirofski, L A; Thomas, E K; Scharff, M D (1993) Variable region gene utilization and mutation in a group of neutralizing murine anti-human immunodeficiency virus type 1 principal neutralizing determinant antibodies. AIDS Res Hum Retroviruses 9:41-9 |
