scholarly journals Transcriptional and Translational Regulation of α-Acetolactate Decarboxylase of Lactococcus lactissubsp. lactis

2000 ◽  
Vol 182 (19) ◽  
pp. 5399-5408 ◽  
Author(s):  
Nathalie Goupil-Feuillerat ◽  
Gérard Corthier ◽  
Jean-Jacques Godon ◽  
S. Dusko Ehrlich ◽  
Pierre Renault
Keyword(s):  
Translational Regulation ◽  
Rna Folding ◽  
Stringent Response ◽  
Second Step ◽  
Stem Loop ◽  
Ribosome Binding ◽  
Branched Chain Amino Acid ◽  
Folding Structure ◽  
Branched Chain ◽  
Favorable Conditions

ABSTRACT The α-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of α-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldBfivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.

scholarly journals Crystal structure and ligand-induced folding of the SAM/SAH riboswitch

2020 ◽  
Author(s):  
Lin Huang ◽  
Ting-Wei Liao ◽  
Jia Wang ◽  
Taekjip Ha ◽  
David M J Lilley
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Translational Regulation ◽  
Stable State ◽  
Helical Structure ◽  
Ribosome Binding Site ◽  
Stem Loop ◽  
Ribosome Binding ◽  
Induced Folding ◽  
Initiation Of Translation

Abstract While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

scholarly journals Branched-chain amino acid and lysine deficiencies exert different effects on mammary translational regulation

2015 ◽  
Vol 98 (11) ◽  
pp. 7846-7855 ◽  
Author(s):  
John Doelman ◽  
Julie J.M. Kim ◽  
Michelle Carson ◽  
John A. Metcalf ◽  
John P. Cant
Keyword(s):  
Amino Acid ◽  
Translational Regulation ◽  
Branched Chain Amino Acid ◽  
Branched Chain

scholarly journals Transcriptional Organization and Posttranscriptional Regulation of the Bacillus subtilis Branched-Chain Amino Acid Biosynthesis Genes

2004 ◽  
Vol 186 (8) ◽  
pp. 2240-2252 ◽  
Author(s):  
Ulrike Mäder ◽  
Susanne Hennig ◽  
Michael Hecker ◽  
Georg Homuth
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Half Life ◽  
Posttranscriptional Regulation ◽  
Full Length ◽  
Loop Structure ◽  
Stem Loop ◽  
Branched Chain Amino Acid ◽  
Stem Loop Structure ◽  
Branched Chain

ABSTRACT In Bacillus subtilis, the genes of the branched-chain amino acids biosynthetic pathway are organized in three genetic loci: the ilvBHC-leuABCD (ilv-leu) operon, ilvA, and ilvD. These genes, as well as ybgE, encoding a branched-chain amino acid aminotransferase, were recently demonstrated to represent direct targets of the global transcriptional regulator CodY. In the present study, the transcriptional organization and posttranscriptional regulation of these genes were analyzed. Whereas ybgE and ilvD are transcribed monocistronically, the ilvA gene forms a bicistronic operon with the downstream located ypmP gene, encoding a protein of unknown function. The ypmP gene is also directly preceded by a promoter sharing the regulatory pattern of the ilvA promoter. The ilv-leu operon revealed complex posttranscriptional regulation: three mRNA species of 8.5, 5.8, and 1.2 kb were detected. Among them, the 8.5-kb full-length primary transcript exhibits the shortest half-life (1.2 min). Endoribonucleolytic cleavage of this transcript generates the 5.8-kb mRNA, which lacks the coding sequences of the first two genes of the operon and is predicted to carry a stem-loop structure at its 5′ end. This processing product has a significantly longer half-life (3 min) than the full-length precursor. The most stable transcript (half-life, 7.6 min) is the 1.2-kb mRNA generated by the processing event and exonucleolytic degradation of the large transcripts or partial transcriptional termination. This mRNA, which encompasses exclusively the ilvC coding sequence, is predicted to carry a further stable stem-loop structure at its 3′ end. The very different steady-state amounts of mRNA resulting from their different stabilities are also reflected at the protein level: proteome studies revealed that the cellular amount of IlvC protein is 10-fold greater than that of the other proteins encoded by the ilv-leu operon. Therefore, differential segmental stability resulting from mRNA processing ensures the fine-tuning of the expression of the individual genes of the operon.

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