Ribosomal Protein Operon Research Articles

Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli

The L11 ribosomal protein operon in Escherichia coli consists of the structural genes for proteins L11 and L1. Hybrid deletion plasmids were constructed carrying these two genes with decreasing amounts of the leader mRNA under lac transcriptional control. Measurements of mRNA and protein synthesis directed by these plasmids in vitro and in vivo demonstrate that the regulation of this operon is post-transcriptional and identifies a region of the mRNA, preceding the proximal L11 gene, important for successful feedback inhibition of L11 and L1 synthesis by L1. Additionally, deletions extending to the ribosome binding site of the L11 gene fail to synthesize both L11 and the downstream L1 protein although synthesis of the corresponding mRNA remains unchanged. These results directly demonstrate the presence of translational coupling in this bicistronic operon.

Transcription of the s10 ribosomal protein operon is regulated by an attenuator in the leader

Previous studies have shown that ribosomal protein L4 specifically inhibits the expression of its own operon, the 11-gene S10 operon. To elucidate the mechanism for this regulation, we have examined the effect of protein L4 on transcription of the S10 operon. Hybridization and gel electrophoresis studies indicate that in the presence of excess L4 only RNA molecules about 140 bases long are transcribed from the S10 operon. These short RNA molecules contain the leader, but not structural gene, sequences. Our results suggest that protein L4 stimulates premature termination (attenuation) of transcription about 30 bases upstream from the start of the first structural gene of the S10 operon. The attenuation appears to be independent of the regulation of translation of the operon. We suggest that attenuation of transcription plays a primary role in the autogenous regulation of the S10 operon.

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.

A secondary promoter for elongation factor Tu synthesis in the str ribosomal protein operon of Escherichia coli.

The str operon of Escherichia coli contains genes for ribosomal proteins S12 and S7 and for elongation factors EF-G and EF-Tu (Jaskunas et al. 1975). We have subcloned various segments of DNA from this operon onto multicopy plasmids. We found that cells carrying a recombinant plasmid which lacks the major promoter for the str operon but contains the 5' portion of the EF-Tu gene synthesize a novel protein which we have identified as a truncated EF-Tu molecule. Moreover, cells carrying plasmids with an intact EF-Tu gene synthesize the elongation factor at a 3- to 5-fold higher rate than haploid cells. Thus the EF-Tu gene can be expressed in the absence of the major promoter for the str operon. This expression is not due to read-through from plasmid promoters, but it is dependent on the presence of the distal portion of the EF-G gene on the plasmids. These results indicate that there is a secondary promoter for EF-Tu expression, apparently located within the structural gene for elongation factor EF-G.

Regulation of the S10 ribosomal protein operon in E. coli: Nucleotide sequence at the start of the operon

We have determined the DNA sequence of a 1250 base pair segment of the Escherichia coli chromosome that carries the promoter for the S10 ribosomal protein operon, the S10 gene and part of the L3 gene. A DNA fragment carrying the putative S10 promoter was cloned into the plasmid mini-Col E1, which contains a transcription termination signal close to the single Hind II site. Cells harboring the hybrid plasmid produced a relatively stable hybrid mRNA with the expected sequence, demonstrating that the promoter functions in vivo. Comparison of the mRNA sequence around the start of the S10 coding region, the presumed target site for L4 repressor protein, with the known binding site for L4 on 23S rRNA has revealed the presence of sequence homologies. This supports the model of the translational feedback regulation of the S10 operon by L4.

An amber mutation in a ribosomal protein gene: ineffective suppression stimulates operon-specific transcription.

The effects of inefficient suppression and the accompanying polarity of an amber mutation in the ribosomal protein gene rplC (L3) on the transcriptional activities of several ribosomal protein operons were investigated. The L3 gene is proximally located in the S10 operon. Inefficient suppression of the mutation specifically stimulated transcription of regions both proximal and distal to the site of mutation by about twofold. In contrast, no effect on the transcriptional activity of regions within the alpha and spc ribosomal protein operons was observed. Thus, transcription of the S10 operon could be actively and specifically regulated in response to a deficiency in one or more of the protein products from the operon. We presume that this transcriptional control acts at the level of transcription initiation, but we cannot exclude the possibility of some other unknown transcriptional regulatory mechanism.

DNA sequence of the promoter region for the alpha ribosomal protein operon in Escherichia coli.

Previous studies showed that the gene for RNA polymerase subunit alpha at 72 min on the Escherichia coli chromosome is co-transcribed with genes for ribosomal proteins (r-proteins) S11, S4, and L17, and probably S13. DNA sequence analysis of a deletion mutant has now established that the S13 gene is a part of the alpha operon and the gene order is promoter (P alpha), rpsM (S13), rpsK (S11), rpsD(S4), rpoA(alpha), and rplQ(L17). The DNA sequence extending 650 bases before S13 gene was determined. In vitro transcription experiments establish the probable location of the alpha promoter (P alpha) within this sequence. The start site is 94 nucleotides upstream from the initiation codon (GUG) of the S13 gene. This promoter has features previously noted as common to E. coli promoters. However, comparison with four other sequenced promoters of r-protein operons shows no unique common features that might account for the common regulation of synthesis of r-proteins. This lack of sequence similarity in promoters of r-protein operons may be because r-protein synthesis is regulated at least partially at a post-transcriptional level.

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable “Pribnow box” sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions −69 through −40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

Isolation and characterization of a promoter mutant in the str ribosomal protein operon in E. coli

The λ fus3 transducing phage carries several operons for ribosomal proteins of E. coli, including the str operon. A mutant transducing phage with a promoter mutation in this operon has been isolated. This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells. This mutation also abolishes in vitro transcription from the str promoter. The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the “Pribnow box” sequence from TAAAATT to TAAAACT. These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon.

Ribosomal RNA synthesis in the F'14 episome-containing minicells of Escherichia coli.

F′14 episome of E. coli, which includes genes for rRNA's but not the “ribosomal protein operon” (Nomura and Engbeak, 1972) has been transferred to a minicell-producing strain of E. coli K12. The RNA formed in the purified episome-containing minicells was identified as rRNA's by sedimentation analyses and DNA-RNA hybridization-competition experiments. It is concluded that the rRNA genes in the F′14 episome can be transcribed without expression of the “ribosomal protein operon”.

The size of transcriptional units for ribosomal proteins in Escherichia coli.

An analysis of the degree to which the 55 ribosomal protein genes are clustered in polycistronic transcriptional units in E. coli is presented. Three kinetic approaches were applied: a) The labeling kinetics of RNA at different times after addition of rifampicin to a culture of E. coli growing in glucose minimal medium was used to calculate the distribution of transcribing RNA polymerases over different size classes of mRNA operons. The average length of transcriptional units being transcribed at a given time is about 3300 base pairs. Less than 1% of mRNA synthesis originates from transcriptional units longer than 12000 base pairs and only about 10% from units longer than 7500 base pairs. From this the upper limit of the length of ribosomal protein operons can be estimated. b) The rate of ribosomal protein synthesis as a fraction of the rate of total protein synthesis (αr) was measured during the cessation of mRNA synthesis and its decay after rifampicin addition. αr appears to decrease from 0.125 to 0.09 indicating that probably most ribosomal protein transcriptional units are shorter than the average. c) The kinetics of the rates of synthesis of individual ribosomal proteins was analysed after the release of the inhibition by rifampicin in a partially rifampicin resistant strain at 40°C. The rates of synthesis for all of the 40 ribosomal proteins tested reach half their final values at times falling in the interval 1.4–3.5 min, though for the majority it was reached between 1.6–2.6 min.