The long term objective of the proposal is to understand at the molecular certain aspects of the synthesis, processing and translation of messenger RNAs in Escherichia coli. Emphasis is placed on identifying regulatory controls that operate during these processes. Coupling of translation between genes V and VII of the filamentous phage will be analyzed, with emphasis on understanding the basis for the severe downregulation at the V- VII intercistronic junction and identifying determinants of coupling efficiency and possible interactions with ribosomal components. The mechanism of translational coupling will be studied in vivo and subsequently in vitro, using a 16S rRNA mutant with an altered anti-Shine- Dalgarno sequence to direct entry of a spectinomycin-resistant ribosome to an upstream cistron bearing the complimentary Shine-Dalgarno sequence. The coupled initiation sites used will be those studied in earlier work which slow no independent activity and function only when immediately downstream of a translated region. The endonucleolytic processing events that generate a number of the major phage f1 mRNAs will be studied further, focusing on the ams/rne-dependent cleavages as likely examples of RNase E cleavage sites. Since production of these RNAs does not occur in temperature-sensitive ams/rne mutants, the regulatory role of processing on phage gene expression or the life cycle can now be explored. As a conclusion to studies using cleavage-defective EcoRI endonuclease mutants as transcriptional roadblocks and probes of elongation complex structure and function, E.coli RNA polymerase movement along the DNA template will be examined.
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