The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes

Zhang, Yian-Biao; Ayalew, Sahlu; Lacks, Sanford A.

doi:10.1128/jb.179.12.3828-3836.1997

Cited by 23 publications

(27 citation statements)

References 41 publications

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“…This sequence was first identified by mutagenesis studies in the mRNA of phage T7 gene 0.3 and subsequently found in the cI, lysU, glnS, rpoH, and cspA mRNAs of Escherichia coli (5)(6)(7)(8)(9)(10). Similar elements have been found in the dnaX gene of Caulobacter crescentus, the vph gene of Streptomyces vinaceus, and the rnhB gene of Streptococcus pneumoniae (11)(12)(13). All of the identified DB elements display partial complementarity to nucleotides 1469-1483 of 16S rRNA.…”

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confidence: 66%

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA

O’Connor

Asai

Squires

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The downstream box (DB) is a sequence element that enhances translation of several bacterial and phage mRNAs. It has been proposed that the DB enhances translation by base pairing transiently to bases 1469-1483 of 16S rRNA, the so-called anti-DB, during the initiation phase of translation. We have tested this model of enhancer action by constructing mutations in the anti-DB that alter its mRNA base-pairing potential and examining expression of a variety of DB-containing mRNAs in strains expressing the mutant anti-DB 16S rRNA. We found that the rRNA mutant was viable and that expression of all tested DB-containing mRNAs was completely unaffected by radical alterations in the proposed anti-DB. These findings lead us to conclude that enhancement of translation by the DB does not involve mRNA-rRNA base pairing.Initiation of translation in bacteria typically involves two base-pairing interactions: the mRNA initiation codon pairs with the anticodon of the initiator methionine tRNA, and the Shine-Delgarno (SD) sequence, 7-10 bases upstream of the initiation codon, pairs with the 3Ј end of 16S rRNA (1). Mutagenesis studies of the initiation signals of particular mRNAs as well as analysis of atypical and leaderless mRNAs have identified other sequence elements surrounding the initiator AUG that contribute to the efficiency of the initiation signal (2). One such class of such elements has been termed translational enhancers, based on their ability to enhance the expression of various mRNA reporter gene constructs or to promote initiation in the absence of any identifiable SD. Among the best-studied enhancer elements is the downstream box (DB), so called because of its location downstream of the AUG initiation codon (3, 4). This sequence was first identified by mutagenesis studies in the mRNA of phage T7 gene 0.3 and subsequently found in the cI, lysU, glnS, rpoH, and cspA mRNAs of Escherichia coli (5-10). Similar elements have been found in the dnaX gene of Caulobacter crescentus, the vph gene of Streptomyces vinaceus, and the rnhB gene of Streptococcus pneumoniae (11-13). All of the identified DB elements display partial complementarity to nucleotides 1469-1483 of 16S rRNA. Moreover, mutagenesis studies have indicated that, in general, increases in the level of complementarity to this region of 16S rRNA led to increased expression whereas mutations that decreased complementarity caused corresponding reductions in expression of the reporter genes. Based on these observation, it has been concluded that DB elements enhance and stabilize the interactions of initiating ribosomes with mRNAs by base pairing to nucleotides 1469-1483 of 16S rRNA, the so-called anti-DB.Although the mRNA mutagenesis studies cited above lend considerable credence to the mRNA-rRNA base-pairing model of enhancer action, supporting biochemical or rRNA mutagenesis data are lacking. Base-pairing interactions between mRNA and rRNA place considerable constraints on the orientation of mRNA within the ribosome. The mRNA path through the ribosome has ...

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mentioning

confidence: 66%

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA

O’Connor

Asai

Squires

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Additionally, the recently published sequence of RNase HII from Staphylococcus pneumoniae (290 aa, see ref. 23) was detected. The authors of that work already provided evidence that bacterial RNase HII is very widely or universally distributed and has a homologous counterpart in human cells.…”

Section: Resultsmentioning

confidence: 95%

“…Much less is known about the second prokaryotic enzyme, RNase HII, which was cloned from E. coli and then was designated a minor activity of unknown function (21). Comparative renaturation gel assay data of E. coli cell extracts (22) and the recently published characterization of the related Streptococcus pneumoniae RNase HII (23) indicate that the importance of these enzymes has been underestimated. This idea recently was corroborated by identification of the homologous gene from the yeast Saccharomyces cerevisiae (24), which in this organism codes for the main RNase H. Here we will present the experimental data for the determination of the complete amino acid sequence of the large subunit of human RNase HI, which will unveil the prokaryotic RNase HII as the evolutionary counterpart of the major mammalian RNase H. We will further show that related ORFs are present in genomes of organisms from all three kingdomes (archaebacteria, eubacteria, and eukaryotes).…”

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confidence: 99%

Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII

Frank

Braunshofer-Reiter

Wintersberger

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS͞PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.

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“…The 5Ј mRNA termini as determined by primer extension were consistent with the transcriptional organization. The three termini were located upstream of ylxM (P1), at the adenosine of the translation start codon of ylxM (P2), and in the satC-satD intergenic region (28,34,53), E. coli and streptomycetes (22). Although leaderless transcripts lack upstream Shine-Dalgarno ribosome binding sites, they are functional in translation.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the sat Operon in Streptococcus mutans : Evidence for a Role of Ffh in Acid Tolerance

et al. 2001

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An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bonafide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3 to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans.Acid tolerance is regarded as an important determinant for survival of cariogenic bacteria in the biofilm on teeth (7,37,49.) The microorganisms in dental plaque are exposed to continual cycles of acid shock resulting from the formation of acid end products during metabolism of dietary carbohydrate by the acidogenic microflora. In vivo pH measurements have revealed that the intake of carbohydrates can drop the plaque pH from 7 to 4 in less than 20 min (21, 24, 52). In addition, frequent carbohydrate intake results in a lower pH of "resting plaque," which has been associated with increased dental caries. Early studies (41) demonstrated that high caries activity is associated with rapid plaque acidification as well as tolerance of low pH by the plaque microorganisms.

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The rnhB gene encoding RNase HII of Streptococcus pneumoniae and evidence of conserved motifs in eucaryotic genes

Cited by 23 publications

References 41 publications

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA

Enhancement of translation by the downstream box does not involve base pairing of mRNA with the penultimate stem sequence of 16S rRNA

Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII

Characterization of the sat Operon in Streptococcus mutans : Evidence for a Role of Ffh in Acid Tolerance

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