Structural probing and mutagenic analysis of the stem-loop required for Escherichia coli dnaX ribosomal frameshifting: programmed efficiency of 50%

ABSTRACT The rare codons AGG and AGA comprise 2% and 4%, respectively, of the arginine codons of Escherichia coli K-12, and their cognate tRNAs are sparse. At tandem occurrences of either rare codon, the paucity of cognate aminoacyl tRNAs for the second codon of the pair facilitates peptidyl-tRNA shifting to the +1 frame. However, AGG_AGG and AGA_AGA are not underrepresented and occur 4 and 42 times, respectively, in E. coli genes. Searches for corresponding occurrences in other bacteria provide no strong support for the functional utilization of frameshifting at these sequences. All sequences tested in their native context showed 1.5 to 11% frameshifting when expressed from multicopy plasmids. A cassette with one of these sequences singly integrated into the chromosome in stringent cells gave 0.9% frameshifting in contrast to two- to four-times-higher values obtained from multicopy plasmids in stringent cells and eight-times-higher values in relaxed cells. Thus, +1 frameshifting efficiency at AGG_AGG and AGA_AGA is influenced by the mRNA expression level. These tandem rare codons do not occur in highly expressed mRNAs.

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Regulation of Expression of the adhE Gene, Encoding Ethanol Oxidoreductase in Escherichia coli: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS

Journal of Bacteriology ◽

10.1128/jb.181.24.7571-7579.1999 ◽

1999 ◽

Vol 181 (24) ◽

pp. 7571-7579 ◽

Cited By ~ 28

Author(s):

Jorge Membrillo-Hernández ◽

E. C. C. Lin

Keyword(s):

Escherichia Coli ◽

Transcriptional Start Site ◽

Start Codon ◽

Start Site ◽

Rnase Iii ◽

Regulation Of Expression ◽

Experimental Conditions ◽

Stem Loop ◽

Translational Start ◽

Transcriptional Start Sites

ABSTRACT The adhE gene of Escherichia coli, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of adhEis dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the lacZ gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activatesadhE transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.

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mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures

Molecular Microbiology ◽

10.1111/j.1365-2958.1994.tb01310.x ◽

1994 ◽

Vol 14 (4) ◽

pp. 731-741 ◽

Cited By ~ 31

Author(s):

Helen Causton ◽

Béatrice Py ◽

Robert S. McLaren ◽

Christopher F. Higgins

Keyword(s):

Escherichia Coli ◽

Mrna Degradation ◽

Stem Loop

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CCC.UGA: a new site of ribosomal frameshifting in Escherichia coli

Gene ◽

10.1016/0378-1119(94)90602-5 ◽

1994 ◽

Vol 143 (1) ◽

pp. 43-47 ◽

Cited By ~ 15

Author(s):

Maarten H. de Smit ◽

Jan van Duin ◽

Peter H. van Knippenberg ◽

Hendrik G. van Eijk

Keyword(s):

Escherichia Coli ◽

Ribosomal Frameshifting

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mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures

Trends in Genetics ◽

10.1016/0168-9525(95)90228-7 ◽

1995 ◽

Vol 11 (4) ◽

pp. 131

Keyword(s):

Escherichia Coli ◽

Mrna Degradation ◽

Stem Loop

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The Bulged Nucleotide in the Escherichia coli Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach

Journal of Bacteriology ◽

10.1128/jb.182.22.6302-6307.2000 ◽

2000 ◽

Vol 182 (22) ◽

pp. 6302-6307 ◽

Cited By ~ 17

Author(s):

Chuang Li ◽

Myriam Reches ◽

Hanna Engelberg-Kulka

Keyword(s):

Escherichia Coli ◽

Insertion Sequence ◽

Stop Codon ◽

Loop Structure ◽

Genetic Approach ◽

Stem Loop ◽

E Coli ◽

Stem Loop Structure ◽

Suppressor Mutations

ABSTRACT The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires acis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. InEscherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of thefdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizingselB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selBsuppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a “single suppressible unit,” suggesting that there is some flexibility to the nature of the interaction between this element and SelB.

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Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

Nucleic Acids Research ◽

10.1093/nar/gkr579 ◽

2011 ◽

Vol 39 (20) ◽

pp. 8952-8959 ◽

Cited By ~ 31

Author(s):

C.-H. Yu ◽

M. H. Noteborn ◽

C. W. A. Pleij ◽

R. C. L. Olsthoorn

Keyword(s):

Stem Loop ◽

Ribosomal Frameshifting ◽

Rna Pseudoknot

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Pleiotropic effects of mutations at positions 13 and 914 in Escherichia coli 16S ribosomal RNA

Biochemistry and Cell Biology ◽

10.1139/o95-098 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 907-913 ◽

Cited By ~ 4

Author(s):

Léa Brakier-Gingras ◽

Robert Pinard ◽

François Dragon

Keyword(s):

Escherichia Coli ◽

Ribosomal Rna ◽

Pleiotropic Effects ◽

16S Ribosomal Rna ◽

Order Structure ◽

Initiation Complex ◽

Stem Loop ◽

Translational Initiation ◽

Slowing Down ◽

Translational Accuracy

Mutations at position 13 or 914 of Escherichia coli 16S ribosomal RNA exert pleiotropic effects on protein synthesis. They interfere with the binding of streptomycin, a translational miscoding drug, to the ribosomes. They increase translational fidelity, and this effect can be related to a perturbation of the higher order structure of the 530 stem–loop, a key region for tRNA selection. In contrast, the structure of the decoding center is not perturbed. The mutations also affect translational initiation, slowing down the formation of the 30S initiation complex. This effect can be related to a destabilization of the pseudoknot helix (17–19/916–918), at the convergence of the three major domains of 16S ribosomal RNA.Key words: ribosomal RNA, translational accuracy, translational initiation.

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