scholarly journals Overexpression of tnaC of Escherichia coli Inhibits Growth by Depleting tRNA2Pro Availability

2006 ◽  
Vol 188 (5) ◽  
pp. 1892-1898 ◽  
Author(s):  
Ming Gong ◽  
Feng Gong ◽  
Charles Yanofsky
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Growth Inhibition ◽  
Leader Peptide ◽  
Multicopy Plasmid ◽  
Coding Region ◽  
Rate Reduction ◽  
Growth Rate Reduction ◽  
Transcription Antitermination ◽  
Sense Codon

ABSTRACT Transcription of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. Induction results from ribosome stalling after translation of tnaC, the coding region for a 24-residue leader peptide. The last sense codon of tnaC, proline codon 24 (CCU), is translated by tRNA2 Pro. We analyzed the consequences of overexpression of tnaC from a multicopy plasmid and observed that under inducing conditions more than 60% of the tRNA2 Pro in the cell was sequestered in ribosomes as TnaC-tRNA2 Pro. The half-life of this TnaC-tRNA2 Pro was shown to be 10 to 15 min under these conditions. Plasmid-mediated overexpression of tnaC, under inducing conditions, reduced cell growth rate appreciably. Increasing the tRNA2 Pro level relieved this growth inhibition, suggesting that depletion of this tRNA was primarily responsible for the growth rate reduction. Growth inhibition was not relieved by overexpression of tRNA1 Pro, a tRNAPro that translates CCG, but not CCU. Replacing the Pro24CCU codon of tnaC by Pro24CCG, a Pro codon translated by tRNA1 Pro, also led to growth rate reduction, and this reduction was relieved by overexpression of tRNA1 Pro. These findings establish that the growth inhibition caused by tnaC overexpression during induction by tryptophan is primarily a consequence of tRNAPro depletion, resulting from TnaC-tRNAPro retention within stalled, translating ribosomes.

scholarly journals Role of Ribosome Release in Regulation oftna Operon Expression in Escherichia coli

1999 ◽  
Vol 181 (5) ◽  
pp. 1530-1536 ◽  
Author(s):  
Kouacou Vincent Konan ◽  
Charles Yanofsky
Keyword(s):  
Escherichia Coli ◽  
Mutant Allele ◽  
Stop Codon ◽  
Leader Peptide ◽  
Coding Region ◽  
Reporter Construct ◽  
E Coli ◽  
Operon Expression ◽  
Basal Level Expression

ABSTRACT Expression of the degradative tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the tna operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region, tnaC, that contains a single, crucial, Trp codon. Studies with a lacZ reporter construct lacking the spacer region between tnaC and the first major structural gene,tnaA, suggested that tryptophan induction might involvecis action by the TnaC leader peptide on the ribosome translating the tnaC coding region. The leader peptide was hypothesized to inhibit ribosome release at thetnaC stop codon, thereby blocking Rho’s access to the transcript. Regulatory studies with deletion constructs of thetna operon of Proteus vulgaris supported this interpretation. In the present study the putative role of thetnaC stop codon in tna operon regulation inE. coli was examined further by replacing the naturaltnaC stop codon, UGA, with UAG or UAA in atnaC-stop codon-tnaA′-′lacZ reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in E. coli translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the tnaC stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1, prfA1, increased basal level expression 60-fold when the tnaC stop codon was UAG and 3-fold when this stop codon was UAA; basal level expression was reduced by 50% in the construct with the natural stop codon, UGA. In strains with any of the three stop codons and the prfA1mutation, the induced levels of tna operon expression were virtually identical. The effects of tnaC stop codon identity on expression were also examined in the absence of Rho action, using tnaC-stop codon-′lacZ constructs that lack the tnaC-tnaA spacer region. Expression was low in the absence of tnaC stop codon suppression. In most cases, tryptophan addition resulted in about 50% inhibition of expression when UGA was replaced by UAG or UAA and the appropriate suppressor was present. Introduction of the prfA1 mutant allele increased expression of the suppressed construct with the UAG stop codon; tryptophan addition also resulted in ca. 50% inhibition. These findings provide additional evidence implicating the behavior of the ribosome translating tnaC in the regulation of tna operon expression.

EVOLUTION OF TRANSPOSONS: NATURAL SELECTION FOR Tn5 IN ESCHERICHIA COLI K12

Genetics ◽  
1983 ◽  
Vol 103 (4) ◽  
pp. 581-592
Author(s):  
Susan Wurster Biel ◽  
Daniel L Hartl
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rate Effect ◽  
Coding Region ◽  
Bacterial Populations ◽  
The Right ◽  
Derivatives Of ◽  
Intact Element ◽  
Prior Adaptation

ABSTRACT A novel in vivo effect of the transposable element Tn5 has been observed in chemostats when certain isogenic Tn5 and non-Tn5 strains of Escherichia coli compete for a limiting carbon source in the absence of kanamycin. The Tn5-bearing strain has a more rapid growth rate and increases in frequency from 50% to 90% within the first 15 to 20 generations. The effect occurs when Tn5 is inserted at a variety of chromosomal locations or when the element is carried by an episome, but it is strain specific, having been observed in two out of three strains examined. (For reasons unknown, the effect has not been observed with derivatives of strain CSH12.) Although the growth-rate advantage of Tn5 is independent of nutrient concentration and generation time, it can be reduced by prior adaptation of the strains to limiting conditions, and the amount of reduction is proportional to the length of prior adaptation. The growth-rate effect is evidently not caused by beneficial mutations induced by Tn5 transposition, as Tn5-bearing strains selected in chemostats retain their initial Tn5 position and copy number. However, the effect does not occur in Tn5-112, a transpositionless deletion mutation missing the transposase-coding region of the right-hand IS sequence flanking the element. Since Tn5-112 retains a functional kanamycin-phosphotransferase gene, this gene is not responsible for the growth-rate effect. Thus, the effect evidently requires transposase function, but it does not involve actual transposition of the intact element. Altogether, these data provide a mechanism for the maintenance of Tn5 in bacterial populations in the absence of kanamycin, and they suggest a model for the proliferation and the maintenance of IS sequences and transposable elements in the absence of other identifiable selection pressures.

Growth Rate Reduction during Energy Restriction in Obese Adolescents

2009 ◽  
Vol 96 (04) ◽  
pp. 73-82 ◽  
Author(s):  
M. Amador ◽  
Lidia T. Ramos ◽  
Marta Moroño ◽  
Mirta P. Hermelo
Keyword(s):  
Growth Rate ◽  
Energy Restriction ◽  
Rate Reduction ◽  
Obese Adolescents ◽  
Growth Rate Reduction

Progressive growth rate reduction due to impurity desorption in vapor grown hexamethylenetetramine crystals

2005 ◽  
Vol 40 (10-11) ◽  
pp. 1076-1081 ◽  
Author(s):  
C. Paorici ◽  
M. Zha ◽  
C. Razzetti ◽  
L. Zanotti ◽  
E. Bassano ◽  
...  
Keyword(s):  
Growth Rate ◽  
Rate Reduction ◽  
Growth Rate Reduction ◽  
Progressive Growth

scholarly journals Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons

1999 ◽  
Vol 181 (12) ◽  
pp. 3803-3809 ◽  
Author(s):  
Tsuneaki Asai ◽  
Ciarán Condon ◽  
Justina Voulgaris ◽  
Dmitry Zaporojets ◽  
Binghua Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rrna Operon ◽  
Rrna Genes ◽  
23S Rrna ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Protein Ratio ◽  
23S Rrna Genes

ABSTRACT The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

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