Portable Shine-Dalgarno Regions: A System for a Systematic Study of Defined Alterations of Nucleotide Sequences within<i>E. coli</i>Ribosome Binding Sites

Boer, H. H.; Hui, Anna; Comstock, Lisa J.; Wong, Edith Y.; Vasser, Mark

doi:10.1089/dna.1983.2.231

Cited by 74 publications

(31 citation statements)

References 22 publications

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“…2A). However, the mutation of an SD sequence in a canonical E. coli mRNA would typically result in complete loss of expression, because the SD sequence is the primary ribosome binding signal (28)(29)(30). Also, in agreement with the expression observed with the ptrB SD sequence mutant (Table 1, pAptrB.SDmut), a 30S subunit binding signal was found to be present at the internal start codon in the primer extension inhibition assay (toeprint assay) (Fig.…”

Section: Figsupporting

confidence: 66%

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Beck

Janssen

2017

J Bacteriol

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Alternative translation initiation mechanisms, distinct from the ShineDalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5= terminus of its mRNA. The 5=-terminal AUG (5=-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5=-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5=-uAUG. Additional sequences within the ptrB 5= untranslated region (5=-UTR) work cumulatively with the 5=-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5=-UTRs from other mRNAs with the ptrB 5=-UTR sequence showed a similar dependence on the 5=-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5=-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5=-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5= termini of E. coli mRNAs and the ability of ptrB 5=-UTR regulation to function independently of gene context, the regulatory effects of 5=-uAUGs on downstream CDSs may be widespread throughout the E. coli genome.IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.KEYWORDS 5= upstream AUG, Shine-Dalgarno, noncanonical initiation, translation initiation, translational regulation, upstream open reading frame P rotein synthesis, in which the ribosome translates an mRNA sequence to form polypeptides, is a four-step process that includes initiation, elongation, termination, and ribosome recycling. Translation initiation is energy dependent and the most highly regulated phase of translation (1). The translational machinery interacts with the mRNA at its ribosome binding site (RBS) along with initiator tRNA to form a ternary complex that is equipped for polypeptide productio...

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Section: Figsupporting

confidence: 66%

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Beck

Janssen

2017

J Bacteriol

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“…(6). These fragments of partially digested chromosomal DNA from Z. mobilis were fused to a truncated E. coli lacZ gene and contained many of the features of E. coli consensus promoter regions (9,12,27 and the translational start (7). The occurrence of TAA immediately downstream from the ribosome-binding region in pdc from Z. mobilis may contribute to its high level of expression in Z. mobilis.…”

Section: Methodsmentioning

confidence: 99%

Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase

Conway¹,

Osman²,

Konnan³

et al. 1987

J Bacteriol

137

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DNA sequence analysis showed that pyruvate decarboxylase (one of the most abundant proteins in Zymomonas mobiis) contains 559 amino acids. The promoter for the gene encoding pyruvate decarboxylase was not recognized by Escherichia coli, although the cloned gene was expressed at relatively high levels under the control of alternative promoters. The promoter region did not contain sequences which could be identified as being homologous to the generalized promoter structure for E. coli. Hydropathy plots for the amino acid sequence indicated that pyruvate decarboxylase contains a large number of hydrophobic domains which may contribute to the thermal stability of this enzyme.The regeneration of NAD+ in the obligately fermentative bacterium Zymomonas mobilis is carried out with two enzymes, pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) (23,30). Although this simple regeneration system is widely utilized by eucaryotes such as the yeasts, fungi, and higher plants (31), it is relatively rare among the bacteria (10, 29). Very few bacterial genera have been shown to contain pyruvate decarboxylase, the key enzyme catalyzing the nonoxidative decarboxylation of pyruvate to produce acetaldehyde plus CO2 In Z. mobilis, this enzyme is reported to be a tetramer of identical subunits with a molecular weight of approximately 55,000 + 5,000 (13). Recent studies by Brau and Sahm (5) have described the cloning of pyruvate decarboxylase from Z. mobilis. These studies indicated that the gene encoding pyruvate decarboxylase is contained on a 4.6-kilobase-pair (kb) SphI fragment.The pyruvate decarboxylase gene is normally expressed at high levels in Z. mobilis and constitutes up to 5% of the total soluble protein (2). Brau and Sahm (5) have also reported a high level of expression of this Z. mobilis gene in Escherichia coli. Previous studies in our laboratory (6; T. Conway and L. 0. Ingram, Abstr. Annu. Meet. Am. Soc. Microbiol. 1986, H62, p. 137; T. Conway and L. 0. Ingram, submitted for publication) defined sequences of DNA fragments from Z. mobilis which exhibited promoter activity in both E. coli and Z. mobilis. These studies indicated that similar regions of DNA were recognized as promoters by both organisms, although the dominant sites of transcriptional initiation were usually different. In this study, we examined the sequence of the pyruvate decarboxylase gene from Z. mobilis and its promoter and identified the sites of transcriptional initiation in both Z. mobilis and E. coli. Our studies indicate that the high level of expression of this gene in E. coli is not due to the recognition of the native Z. mobilis promoter. MATERIAL AND METHODSBacterial strains, plasmids, and growth conditions. The strains and plasmids used in this study are summarized in (20,000 lb/in2). Cell debris was removed by centrifugation for 30 min at 20,000 x g at 4°C. Pyruvate decarboxylase (120 mg) was purified from the resulting soluble protein fraction essentially as described by Hoppner and Doelle (13). The final yields ...

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“…To overcome this dilemma and to facilitate future structure and function studies of any essential region in the 16S rRNA by mutational analysis, we developed the specialized ribosome system (de Boer et al, 1985(de Boer et al, , 1986. In this system the anti Shine -Dalgarno (ASD) sequence near the 3' end of the 16S rRNA molecule was altered.…”

Section: Introductionmentioning

confidence: 99%

Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

Hui¹,

Eaton²,

Boer³

1988

The EMBO Journal

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In the specialized ribosome system, a distinct pool of mutated ribosomes is dedicated to the translation of one particular mRNA species. This was accomplished by altering the Shine-LDalgarno sequence on the mRNA and its complementary anti-Shine -Dalgarno sequence on the plasmid-borne 16S rRNA gene. Here, using the specialized ribosome system, we were able to introduce mutations in key regions of the 16S rRNA and could study their effect on translation in vivo. The C 1400 region has been implicated to play a role in the actual mRNA decoding process. Several ribosomal mutations were introduced in this region. We showed that substitution of the evolutionary highly conserved C1400 residue by a G-or an A-residue inhibits ribosomal activity by 80% and 50% respectively, whereas, a C to a U change at this conserved position does not affect overall ribosomal activity. The adjacent stem structure (1410-1490) was also examined. Disruption of the stem by replacing either one of the arms of this stem, with a different sequence, inhibits ribosomal activity by -80%. A small but significant restoration of translation could be achieved by recreating a complementary stem with a different sequence. We found that full reversion of activity could be obtained when such mutated ribosomes were made spectinomycin resistant by introducing a C to A substitution at position 1192 which is located far away in the secondary structure map of the 16S rRNA molecule. Based on these results we conclude that some, but not all, of the nucleotides in the conserved C1400 region play a key role in translation. Most importantly, we show that the specialized ribosome system provides a very powerful tool for structure and function studies of any region in the complex ribosomal RNA molecule.

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Portable Shine-Dalgarno Regions: A System for a Systematic Study of Defined Alterations of Nucleotide Sequences withinE. coliRibosome Binding Sites

Cited by 74 publications

References 22 publications

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase

Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

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