An archaebacterial RNA polymerase binding site and transcription initiation of the<i>his</i>A gene in<i>Methanococcus vannielii</i>

Brown, James W.; Thomm, Michael; Beckler, Gregory S.; Frey, Gerhard; Stetter, Karl O.; Reeve, John N.

doi:10.1093/nar/16.1.135

Cited by 50 publications

(34 citation statements)

References 17 publications

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“…Primer extension reactions containing 40 g of RNA and 2 pmol of end-labelled lacZ specific oligonucleotide were performed as described previously (Brown et al, 1988). Equal volumes of each reaction were electrophoresed against appropriate dideoxy sequencing reactions, and visualized by autoradiography.…”

Section: Primer Extension Analysismentioning

confidence: 99%

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Etten

Janssen

1998

Molecular Microbiology

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SummaryWe determined the in vivo translational efficiency of 'unleadered' lacZ compared with a conventionally leadered lacZ with and without a Shine-Dalgarno (SD) sequence in Escherichia coli and found that changing the SD sequence of leadered lacZ from the consensus 5Ј-AGGA-3Ј to 5Ј-UUUU-3Ј results in a 15-fold reduction in translational efficiency; however, removing the leader altogether results in only a twofold reduction. An increase in translation coincident with the removal of the leader lacking a SD sequence suggests the existence of stronger or novel translational signals within the coding sequence in the absence of the leader. We examined, therefore, a change in the translational signals provided by altering the AUG initiation codon to other naturally occurring initiation codons (GUG, UUG, CUG) in the presence and absence of a leader and find that mRNAs lacking leader sequences are dependent upon an AUG initiation codon, whereas leadered mRNAs are not. This suggests that mRNAs lacking leader sequences are either more dependent on perfect codon-anticodon complementarity or require an AUG initiation codon in a sequence-specific manner to form productive initiation complexes. A mutant initiator tRNA with compensating anticodon mutations restored expression of leadered, but not unleadered, mRNAs with UAG start codons, indicating that codon-anticodon complementarity was insufficient for the translation of mRNA lacking leader sequences. These data suggest that a cognate AUG initiation codon specifically serves as a stronger and different translational signal in the absence of an untranslated leader.

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Section: Primer Extension Analysismentioning

confidence: 99%

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Etten

Janssen

1998

Molecular Microbiology

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“…The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with 0-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90°C), thermostable, and resistant to common (7,34). Taken together, these data suggest that transcriptional apparatus has been specifically conserved between archaebacteria and eukaryotes.…”

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confidence: 84%

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Because of the extreme growth conditions of archaebacteria, in most cases it is difficult both to obtain amounts of enzymes needed for structure-function studies and to devise methodologies for genetic and molecular studies of regulatory mechanisms.

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mentioning

confidence: 99%

Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression

et al. 1992

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The lacS gene from the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus encodes an enzyme with 0-galactosidase activity that, like other enzymes from this organism, is exceptionally thermophilic (optimal activity above 90°C), thermostable, and resistant to common (7,34). Taken together, these data suggest that transcriptional apparatus has been specifically conserved between archaebacteria and eukaryotes.Because of the extreme growth conditions of archaebacteria, in most cases it is difficult both to obtain amounts of enzymes needed for structure-function studies and to devise methodologies for genetic and molecular studies of regulatory mechanisms. Expression of archaebacterial genes in mesophilic hosts could help to overcome in part these problems, allowing comparative studies of the structure of genes and their regulatory regions and the attainment of large quantities of enzymes by using standard fermentation methods. In this study, we undertook the expression of a thermostable ,B-galactosidase from S. solfataricus in a wellcharacterized host for heterologous gene expression, Saccharomyces cerevisiae.The S. solfataricus ,-galactosidase, which has been purified to homogeneity and biochemically characterized (31), is a 240-kDa tetramer whose mechanism of reaction is similar to those of well-known P-galactosidases from mesophilic organisms but which is distinctive in its thermostability and thermophilicity. The lacS gene encoding this enzyme (8) fails to show significant sequence homology with the lacZ gene of E. coli or other known P-galactosidase-encoding genes.Besides the obvious biotechnological value due to its peculiarities, this enzyme is an interesting model for the study of thermostable enzymes.

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“…Primer extension reactions containing 60 mg of in vivo RNA or 1 mg of in vitro synthesized RNA and 2 pmol of an endlabeled lacZ-specific oligoucleotide were performed as described previously (Brown et al 1988). The primer extension reactions were subjected to electrophoresis against appropriate dideoxy sequencing reactions and visualized by autoradiography.…”

Section: Rna Isolation and Primer Extension Analysismentioning

confidence: 99%

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

Giliberti¹,

O’Donnell²,

Etten³

et al. 2012

RNA

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The bacteriophage l's cI mRNA was utilized to examine the importance of the 59-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 59-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 59-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 59-phosphate or a 59-hydroxyl indicate that leaderless cI mRNA requires a 59-phosphate for stable ribosome binding in vitro as well as expression in vivo. Ribosome-binding assays show that 30S subunits and 70S ribosomes do not bind as strongly to 59-hydroxyl as they do to 59-phosphate containing leaderless mRNA and the tRNAdependent ternary complex is less stable. Additionally, filter-binding assays revealed that the 70S ternary complex formed with a leaderless mRNA containing a 59-hydroxyl has a dissociation rate (k off ) that is 4.5-fold higher compared with the complex formed with a 59-phosphate leaderless mRNA. Fusion to a lacZ reporter gene revealed that leaderless cI mRNA expression with a 59-hydroxyl was >100-fold lower than the equivalent mRNA with a 59-phosphate. These data indicate that a 59-phosphate is an important feature of leaderless mRNA for stable ribosome binding and expression.

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An archaebacterial RNA polymerase binding site and transcription initiation of thehisA gene inMethanococcus vannielii

Cited by 50 publications

References 17 publications

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

Expression of the thermostable beta-galactosidase gene from the archaebacterium Sulfolobus solfataricus in Saccharomyces cerevisiae and characterization of a new inducible promoter for heterologous expression

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

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