scholarly journals CsrA Inhibits Translation Initiation of Escherichia coli hfq by Binding to a Single Site Overlapping the Shine-Dalgarno Sequence

2007 ◽  
Vol 189 (15) ◽  
pp. 5472-5481 ◽  
Author(s):  
Carol S. Baker ◽  
Lél A. Eöry ◽  
Helen Yakhnin ◽  
Jeffrey Mercante ◽  
Tony Romeo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Translation Initiation ◽  
Binding Sites ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Rnase E ◽  
Mobility Shift ◽  
Ribosome Binding ◽  
Shine Dalgarno Sequence

ABSTRACT Csr (carbon storage regulation) of Escherichia coli is a global regulatory system that consists of CsrA, a homodimeric RNA binding protein, two noncoding small RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering this protein, and CsrD, a specificity factor that targets CsrB and CsrC for degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and pgaA by binding to their leader transcripts and preventing ribosome binding. Translation inhibition is thought to contribute to the observed mRNA destabilization. Each of the previously known target transcripts contains multiple CsrA binding sites. A position-specific weight matrix search program was developed using known CsrA binding sites in mRNA. This search tool identified a potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This putative CsrA binding site matched the SELEX-derived binding site consensus sequence in 8 out of 12 positions. Results from gel mobility shift and footprint assays demonstrated that CsrA binds specifically to this site in the hfq leader transcript. Toeprint and cell-free translation results indicated that bound CsrA inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of csrA caused elevated expression of an hfq′-′lacZ translational fusion, while overexpression of csrA inhibited expression of this fusion. We also found that hfq mRNA is stabilized upon entry into stationary-phase growth by a CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first example of a CsrA-regulated gene that contains only one CsrA binding site.

scholarly journals CsrA Regulates Translation of the Escherichia coli Carbon Starvation Gene, cstA, by Blocking Ribosome Access to the cstA Transcript

2003 ◽  
Vol 185 (15) ◽  
pp. 4450-4460 ◽  
Author(s):  
Ashok K. Dubey ◽  
Carol S. Baker ◽  
Kazushi Suzuki ◽  
A. Daniel Jones ◽  
Pallavi Pandit ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genomic Sequence ◽  
Carbon Starvation ◽  
Receptor Protein ◽  
Peptide Transporter ◽  
Wild Type ◽  
Mobility Shift ◽  
Ribosome Binding ◽  
Mutant Strains ◽  
Shine Dalgarno Sequence

ABSTRACT CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA′-′lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Εσ70. We investigated the influence of σS on cstA expression and found that a σS deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.

Translation initiation in Escherichia coli: sequences within the ribosome-binding site

1992 ◽  
Vol 6 (9) ◽  
pp. 1219-1229 ◽  
Author(s):  
Steven Ringquist ◽  
Sidney Shinedling ◽  
Doug Barrick ◽  
Louis Green ◽  
Jonathan Binkley ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Translation Initiation ◽  
Ribosome Binding Site ◽  
Ribosome Binding

scholarly journals Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Hongmarn Park ◽  
Louise C. McGibbon ◽  
Anastasia H. Potts ◽  
Helen Yakhnin ◽  
Tony Romeo ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Translation Initiation ◽  
Binding Sites ◽  
Sigma Factor ◽  
Rna Structure ◽  
Rna Binding ◽  
Open Reading Frame ◽  
Translational Coupling ◽  
Reading Frame ◽  
Ribosome Binding

ABSTRACT CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli . Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay. IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD , which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD , causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.

scholarly journals Bacillus subtilis LmrA Is a Repressor of the lmrAB and yxaGH Operons: Identification of Its Binding Site and Functional Analysis of lmrB and yxaGH

2004 ◽  
Vol 186 (17) ◽  
pp. 5640-5648 ◽  
Author(s):  
Ken-ichi Yoshida ◽  
Yo-hei Ohki ◽  
Makiko Murata ◽  
Masaki Kinehara ◽  
Hiroshi Matsuoka ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Multidrug Resistance ◽  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Consensus Sequence ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Genome Wide

ABSTRACT The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.

scholarly journals Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

1993 ◽  
Vol 13 (9) ◽  
pp. 5805-5813 ◽  
Author(s):  
M M Wang ◽  
R Y Tsai ◽  
K A Schrader ◽  
R R Reed
Keyword(s):  
Signal Transduction ◽  
Dna Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Initiation Site ◽  
Olfactory Neuron ◽  
Promoter Regions ◽  
Transcriptional Initiation ◽  
Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

scholarly journals Tuning a bi-enzymatic cascade reaction in Escherichia coli to facilitate NADPH regeneration for ε-caprolactone production

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Jinghui Xiong ◽  
Hefeng Chen ◽  
Ran Liu ◽  
Hao Yu ◽  
Min Zhuo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Whole Cell ◽  
Cyclohexanone Monooxygenase ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Whole Cell Biocatalyst ◽  
Substrate Tolerance

Abstractε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.

Mutational and Structural Analysis of the RNA Binding Site for Escherichia coli Ribosomal Protein S7

1994 ◽  
Vol 244 (1) ◽  
pp. 74-85 ◽  
Author(s):  
François Dragon ◽  
Catherine Payant ◽  
Léa Brakier-Gingras
Keyword(s):  
Escherichia Coli ◽  
Structural Analysis ◽  
Ribosomal Protein ◽  
Binding Site ◽  
Rna Binding

scholarly journals Evidence for Context-Dependent Complementarity of Non-Shine-Dalgarno Ribosome Binding Sites to Escherichia coli rRNA

2013 ◽  
Vol 8 (5) ◽  
pp. 958-966 ◽  
Author(s):  
Pamela A. Barendt ◽  
Najaf A. Shah ◽  
Gregory A. Barendt ◽  
Parth A. Kothari ◽  
Casim A. Sarkar
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Context Dependent

DNA-binding and transcriptional regulatory properties of hepatic leukemia factor (HLF) and the t(17;19) acute lymphoblastic leukemia chimera E2A-HLF

1994 ◽  
Vol 14 (9) ◽  
pp. 5986-5996
Author(s):  
S P Hunger ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Lymphoblastic Leukemia ◽  
Consensus Sequence ◽  
Reporter Genes ◽  
Wild Type ◽  
Basic Leucine Zipper

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

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