R-protein Genes Research Articles

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.

Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli

The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate.

In vivo transcription of E. coli genes coding for rRNA, ribosomal proteins and subunits of RNA polymerase: Influence of the stringent control system

The synthesis of RNA and protein components of the ribosome is subject to stringent control. Elicitation of stringent or relaxed control occurs following limitation of aminoacylated tRNA in isogenic relA+ and relA- strains respectively. We examined the transcription of rRNA genes and of three additional mRNA producing segments of the bacterial chromosome. These segments code respectively for (i) 15 ribosomal proteins (r-protein) and the α subunit of RNA polymerase, (ii) four 50S r-proteins and (iii) the β and β′ subunits of RNA polymerase. The first segment is located near 72 min whereas the second and third segments are contiguous with each other and located near 88 min. We have shown that when the relA+ strain was limited for valyl tRNA the unusual nucleotide guanosine tetraphosphate (ppGpp) accumulates and transcription of genes coding for rRNA and r-proteins was reduced; transcription of the genes coding for the β and β′ subunits of RNA polymerase was not affected. When we limited the relA- strain ppGpp failed to accumulate and the transcription of rRNA and r-protein genes was enhanced; in contrast transcription of the β and β′ genes was not enhanced. We have concluded that the stringent control system directly or indirectly regulates the genes coding for RNA and protein components of the ribosome at the level of transcription. The influence of this control system on the transcription of the two distinct and separate clusters of r-protein genes was identical. In addition we have shown that transcription of the genes for the β and β′ subunits of RNA polymerase was distinctly different from transcription of r-protein genes and not subject to stringent control.

Regulation of the expression of ribosomal protein genes in Escherichia coli

The in vivo expression of genes coding for ribosomal proteins has been examined by using a quantitative RNA-DNA hybridization assay. Total cellular RNA isolated from exponential-phase cultures of Escherichia coli growing at rates between 0·67 and 2·10 doublings per hour and from cultures following a nutritional shift-up was hybridized to DNA from a λ transducing phage carrying at least 22 ribosomal protein genes. These r-protein genes code for about 400,000 daltons of protein or about half of the total protein per ribosome. The results indicate that the fraction of the total cellular RNA which is homologous to the r-protein genetic region on the transducing phage, s , increases with increasing growth rate such that s = 0·0016 μ + 0·0006, where μ is the steady-state growth rate in doublings per hour. Comparison of the rate of synthesis of the r-proteins coded for by genes on the phage and the level of r-protein messenger RNA homologous to these genes during steady-state and transition conditions indicates that the expression of r-protein genes is controlled primarily by a mechanism which regulates the amount of mRNA for r-protein which is available for translation. This control may be exercised either at the level of initiation of transcription at the promotors for r-protein genes or at the level of the stability of the r-protein mRNA. The results further suggest the existence of a second control mechanism which influences the translation of r-protein mRNA.

Gene activities for ribosomal components in Escherichia coli B/r.

The relative transcriptional activities of genes coding for ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) at a steady-state growth rates ranging from 0.65 to 2.1 doublings/h can be estimated from previous measurements of the synthesis rates of stable and unstable RNA (Pato & von Meyenburg, 1970; Nierlich, 1972a,b; Bremer et al., 1973; Dennis & Bremer, 1973b, 1974b) and ribosomal proteins (Schleif, 1967; Dennis & Bremer, 1974a). Comparison of these transcriptional activities suggests that the expression of the r-protein genes and rRNA genes is controlled seperately.

Stringent control of the transcriptional activities of ribosomal protein genes in E. coli.

The stringent control system regulates the expression of not only rRNA genes but also r-protein genes. The regulation of r-protein genes takes place by a mechanism which determines the amount of r-protein mRNA available for translation. Presumably this mechanism operates at the level of initiation of transcription at r-protein gene promoters.

Regulation of ribosomal protein synthesis in Escherichia coli B/r.

The differential synthesis rate of ribosomal protein (r-protein), alpha-r (synthesis rate of r-protein divided by synthesis rate of total protein), was measured during the cell division cycle. It was observed that alpha-r remained essentially constant and was not measurably affected by duplication of the r-protein gene cluster (i.e., str-spc region) during the process of chromosome replication. It was further observed that the rate of total protein synthesis and r-protein synthesis increased continuously and uniformly during the entire cell cycle. This gene dosage independence of the synthesis rate of r-protein was similar to that observed earlier for the synthesis of ribosomal ribonucleic acid (rRNA). These observations indicate that the synthesis rates of the protein and RNA components of the ribosome are coordinately balanced during the entire cell division cycle and are not significantly perturbed by duplication of the r-protein or rRNA genes. Furthermore, this balanced synthesis insures that neither free rRNA nor free r-protein accumulate in appreciable amounts during balanced growth.