Ribosomal Protein-RNA Polymerase Operon of E. coli

Niels P. Fiil University institute of Microbiology Oster Farimagsgade 2A, DK 1350 Copenhagen, Denmark James D. Friesen Department of Biology York University 4700 Keele St. Downsview Ontario M3J IP3, Canada Willa Lee Downing Department of Biochemistry University of British Columbia Vancouver British Columbia V6T IW5, Canada Patrick P. Dennis Department of Biochemistry University of British Columbia Vancouver British Columbia V6T IW5, Canada Summary A high copy number plasmid that carries the pro- moter PJ, intact rp/J and a deletion of the 3’terminal portion of rplL is detrimental to the growth of the host bacterium. Six independent point mutations on the plasmid that overcome this detriment have been isolated. Nucleotide sequence analysis demon- strates that all six mutants are single base pair alterations, occur within the leader region of the rp/J operon and are well removed from the pre- sumed position of the primary promoter, P.,. These mutant plasmids exhibit normal transcription of rp/J-rp/L but do not translate rplJ messenger RNA to yield plasmid-specified LlO ribosomal protein. We suggest that these mutations define a regula- tory region within the leader sequence of the RNA transcript that serves to modulate the translational efficiency of rplJ messenger RNA. Introduction The region of 88 min on the E. coli genetic map (Bachmann et al., 1976) contains a cluster of genes specifying four 50s ribosomal proteins and two sub- units of RNA polymerase. The genetic organization and the regulation of these genes is complex. The genes are organized in two separate transcription units as follows: Pk (promoter), rp/K (Ll 1) rp/A (Ll); PJ, (promoter), rp/J (LlO), rp/L (L7/L12), rpoB (p), rpoC operon also appears to have at least one secondary promoter (Newmann, Linn and Hayward, 1979; Holowachuk, Friesen and Fiil, 1980; Friesen et al., 1980) and a transcription attenuator between rplL and rpoB (Dennis, 1977; Linn and Scaife, 1978; Den- nis and Fiil, 1979; Barry, Squires Squires, Newman et al., 1979; Friesen et al., 1980). The atten- uator is responsible for modulating the transcription of the distal rpoBC genes (R. Little and P. P. Dennis, manuscript submitted). Moreover, the second gene of the operon, rp/L, is expressed 4 fold more frequently than is the first gene, rp/J, and other ribosomal pro- teins (Subramanian, 1975). Nucleotide sequence analysis of this operon indicates that there is a long leader sequence of almost 400 bp separating the primary promoter, PJ, from the start of the first struc- tural gene of the operon, rp/J (Post et al., 1979). It seems probable that this region contains important regulatory sequences. It has generally been observed that the presence on high copy number plasmids of ribosomal protein struc- tural genes, along with their controlling sequences, is detrimental to the growth of host bacterium. Pre- sumably, cellular control mechanisms are unable to cope with the imbalance in the production of ribosomal proteins that results from the selective gene amplifi- cation. A notable exception to these observations has been the ability of cells to tolerate high copy number plasmids that carry the rpN operon (Fiil et al., 1979). The reasons for this tolerance remain unknown, al- though recent experiments suggest that strict regula- tory mechanisms operate at both the transcriptional and post-transcriptional levels to limit the expression of the amplified genes and thus to maintain a balance in the production of ribosomal proteins (Dennis and Fiil, 1979; Friesen et al., 1980; Fiil, Friesen and Den- nis, 1980; R. Little and P.P. Dennis, manuscript sub- mitted). In this paper we describe a series of mutants that alter one of these regulatory mechanisms, which appears responsible for modulating the trans- lation of rp/J messenger RNA. Results Genetic Organization of the rif Region Figure 1 shows the genetic organization of the rif region’ near 88 min on the bacterial chromosome (Bachmann, Low and Taylor, 1976) carried on lambda drif? 8 (Kirschbaum and Konrad, 1973). rpoB and rpoC, the structural genes for RNA polymerase subunits /3 and p’, are located at the distal end of a four gene operon that also includes rplJ and rplL, structural genes for ribosomal proteins LIO and L7/ L12. Nearby is another related operon that has two genes, rp/K and rp/A, the structural genes for ribo- somal proteins Lll Ll. The positions of the primary and secondary promoters the attenuator are indicated (Linn and Scaife, 1978; Yamamoto Nomura, 1979; Fiil et al., 1979; Post et al., 1979; Friesen et al., 1980). Two plasmids carrying portions