Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli

Sor, Frédéric; Bolotin‐Fukuhara, Monique; Nomura, Masayasu

doi:10.1128/jb.169.8.3495-3507.1987

Cited by 38 publications

(18 citation statements)

References 45 publications

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“…A predicted RNA secondary structure (29) for the gene X-secA intergenic region (Fig. 5A) shows that the secA ribosome-binding site is occluded by an RNA stem which would be destabilized by a ribosome bound (1,8,15,26). A major difference between these two structures is that the rplO-secY intergenic spacing is only 7 nucleotides long, as opposed to 61 nucleotides for the comparable gene X-secA intergenic region.…”

Section: Discussionmentioning

confidence: 99%

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli

Schmidt

Oliver

1989

J Bacteriol

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The Escherichia coli secA gene, whose expression is responsive to the protein secretion status of the cell, is the second gene in an operon. We found that both the basal and induced levels of SecA biosynthesis are dependent on prior translation of the upstream gene, gene X, and identified two large gene X-secA transcripts. The 10-fold derepression of secA expression by protein export defects was at the translational level since no further increases in gene X or secA mRNA levels were detected during this period, and a secA-lacZ protein fusion but not an operon fusion was appropriately derepressed. Furthermore, overexpression of the SecA protein severely reduced expression of only the secA-lacZ protein fusion, indicating that SecA autogenously represses its own translation.A genetic approach was used to study the molecular mechanisms responsible for protein localization in Escherichia coli. Genetic selections identified a set of sec genes whose products are required to promote the secretion of cell envelope proteins. Conditional lethal secA (13, 18), secY (25), secD (9), and secE (21) mutants have been described which, when shifted to the nonpermissive temperature, accumulate unsecreted protein precursors for most envelope proteins. A nonconditional secB null mutant exhibiting severe export defects for only certain envelope proteins has been described also (11). It has been noted previously that expression of the secA gene is somehow coordinated with the protein secretion status of the cell, since SecA protein synthetic levels are elevated 10-to 20-fold when protein export is blocked in secA, secD, and secY mutants and in strains producing high levels of an export-defective hybrid protein between maltose-binding protein and I3-galactosidase (MalE-LacZ), but not in a secB null mutant (9,19,22). The secA gene is the second gene in an operon in which it is downstream of a gene with an unknown function termed gene X (3, 24). To begin to understand how secA expression is coordinated with the secretion proficiency of the cell, we investigated the regulation of the gene X-secA operon. Our results indicate that secA expression is translationally coupled to gene X and that secA derepression by protein export defects occurs at the translational level. Furthermore, we found that SecA protein autogenously represses its translation. Since a second sec gene, secY, is a distal gene in a ribosomal protein operon and its expression is also translationally coupled (1,8,15), the coregulation of protein secretion with the translational machinery warrants further consideration. MATERIALS AND METHODS

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Section: Discussionmentioning

confidence: 99%

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli

Schmidt

Oliver

1989

J Bacteriol

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“…However, the coupling does not assure strict proportionality between the synthesis of S10 and L3, evidenced both by the 15 to 20% residual translation of the L3 gene during blocked S10 synthesis and by the near-normal rates of L3 translation observed when the translation efficiency of the S10 gene was still only about half the optimal level. Note that the coupling between S10 and L3 is considerably less efficient than the coupling in the two-gene Lll-Li r-protein operon (3,27).…”

Section: Discussionmentioning

confidence: 99%

Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli

et al. 1989

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We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon. We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3. All of the mutations inhibiting S10 synthesis also affected the synthesis of L3. However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain. To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase. As expected, the T7 RNA polymerase was not subject to transcription polarity. Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis. Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.The majority of the ribosomal protein (r-protein) genes of Escherichia coli are organized in multicistronic transcription units (14). The translation of most of these r-protein operons is under autogenous control. That is, one of the r-proteins encoded by a given transcription unit functions as a repressor of the translation of most or all genes in the unit (14,22).The S10 operon codes for 11 r-proteins. Translation of this r-protein operon is regulated by L4, the product of the third gene of the operon (9, 31). Genetic analysis has shown that the target for translation control of the S10 operon is located in the leader (9; L. P. Freedman, Ph.D. thesis, University of Rochester, Rochester, N.Y., 1985). This suggests that, whereas the first gene of the operon is under direct translational inhibition by L4, the regulation of the downstream genes is due to some indirect mechanism, analogous to what has been shown for several other r-protein operons (17,30,32).It has been proposed that the propagation of translational regulation down an r-protein operon depends on translational coupling (22,32), a phenomenon first demonstrated in the tryptophan operon by Oppenheim and Yanofsky (23). When two genes are translationally coupled, efficient translation of a downstream gene depends on the translation of the adjacent upstream gene. Translationally coupled genes are often separated by intercistronic regions which are 3 bases or less (2-4, 23, 25); in several of these cases, the termination codon of the upstream gene even overlaps with the initiation codon of the downstream gene. It has been speculated that the short distance between the genes is essential for efficient translational coupling (23), although coupling has been reported in several r-protein operons which contain genes separated by longer intercistronic regions (17, 30).We have investigated the extent of coupling betw...

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“…Two main views have been put forward (11,24,33,35). One suggests that there is a weak ribosomebinding site for the downstream gene that is aided in translation initiation by ribosomes terminating nearby.…”

Section: Discussionmentioning

confidence: 99%

“…(In this paper, we use the phrase "n bases upstream [or downstream]" to indicate that n bases separate the stop and start codons.) Proximity of translational termination and initiation signals, including overlapping signals, has been described as a feature in coupling translation of adjacent genes (11,24,33,35). Translational coupling has been reported for a number of gene pairs, including galK-galT (29), trpD-trpE (26), and trpB-trpA (1,9).…”

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confidence: 99%

Translational coupling between the ilvD and ilvA genes of Escherichia coli

et al. 1988

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The hypothesis that translation Qf the ilvD and ilvA genes of Escherichia coli may be linked has been examined ip strains in which lacZ-ilvD protein fusions are translated in all three reading frames with respect to ilvD. In these strains, the nucleotide sequence was altered to obtain premature termination of ilvD translation, and in one strain translation termination of ilvO DNA occurred two bases downstream of the ilvA initiation codon. In the wild-type strain, the ilvD translation termination site was located two bases upstream of the ilvA start codon. In each of the mutant strains, expression of ilvA, as determined by the level of threonine deaminase activity, was strikingly lower than in the wild-type strain. The data suggest that expression of ilvD and ilvA is translationally coupled. By inserting a promoterless cat gene downstream of ilvA, it was shown that the differences in enzyme activity were not the result of differences in the amount of ilvA mRNA produced.In members of the family Enterobacteriaceae, the biosynthesis of isoleucine and valine occurs by parallel pathways with enzymes specified by a cluster of genes, ilvGMEDA YC (39). The first five genes in the cluster form the ilvGMEDA operon; ilvY and ilvC are separate transcriptional units. Blazey and Burns (4) first implicated regulation at the translational level in the ilv operon and raised the possibility of translational coupling. Their experiments showed that TnJO insertions in both orientations in the ilvD gene of Salmonella typhimurium exerted an absolute polarity on ilvA expression. In Escherichia coli, a similar strong polar effect has been demonstrated in strains containing bacteriophage Mu-1 insertions (32) or Tn5 insertions (3). Even earlier, it had been noted that the ilvlI188 ochre mutation resulted in a 60% reduction in threonine deaminase activity, although at the time, the effect was thought to be "antipolar" (42).Nucleotide s'equence analysis has revealed that the ilvD translation stop codon and'ilvA start codon are separated by 2 bases, TAA-TA-ATG (8, 18). (In this paper, we use the phrase "n bases upstream [or downstream]" to indicate that n bases separate the stop and start codons.) Proximity of translational termination and initiation signals, including overlapping signals, has been described as a feature in coupling translation of adjacent genes (11,24,33,35). Translational coupling has been reported for a number of gene pairs, including galK-galT (29), trpD-trpE (26), and trpB-trpA (1, 9). In this paper we present evidence that efficient translation of the downstream ilvA gene is dependent on translation of the upstream ilvD gene. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study and their sources are listed in Table 1. The preparation of some of the strains requires special mention. Strain CU1644 was prepared by replacing the wild-type ilv region of CU1318 with that in pPU316, which carries AU(i'G-Y)2243::aad'. The aad+ gene, inserted at the site of the ilv deletion, was derived f...

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Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli

Cited by 38 publications

References 45 publications

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli

SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli

Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli

Translational coupling between the ilvD and ilvA genes of Escherichia coli

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