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Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog.

Willkomm DK, Minnerup J, Hüttenhofer A, Hartmann RK - Nucleic Acids Res. (2005)

Bottom Line: The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus.We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches.By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

View Article: PubMed Central - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie Marbacher Weg 6, D-35037 Marburg, Germany.

ABSTRACT
By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its gamma-proteobacterial homologs (approximately 185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the gamma-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

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5′-RACE analysis of A.aeolicus 6S RNA transcripts. (A) Agarose gel analysis of 5′-RACE products using A.aeolicus total RNA and 6S RNA-specific primers for reverse transcription and PCR. M1, M2, DNA molecular weight markers, with the length (in bp) indicated on the left. −TAP, 5′-RACE without TAP treatment; +TAP, 5′-RACE including TAP digestion, additionally yielding product II which is inferred to represent a primary transcript with a 5′-triphosphate end. Controls: no RT, PCR of total RNA without a prior reverse transcription step; no template, PCR without template. (B) A.aeolicus DNA sequence of the 6S RNA coding region. Bold letters, reading frames pheT (β subunit of phenylalanyl-tRNA synthetase; stop codon marked by the gray box) and aq_1731 (5-formyltetrahydrofolate cyclo-ligase homolog; start codon indicated by gray oval). Small letters, putative 6S RNA coding region; for the 3′ boundary, see Figure 3. Nucleotides identified as 5′ ends of RNAs that yielded prominent bands in the RACE experiment are indicated by arrows with the roman number corresponding to the respective band on the gel in (A) (II, IV: each identified in 4 out of 4 sequenced clones; III, V: each arrow represented by 2 out of 4 sequenced clones); the 5′ end of product I was not determined. Horizontal solid arrow, position of oligonucleotide used as reverse transcription primer and northern probe (Figure 2C); horizontal broken arrow, 3′ primer for the final PCR step of the 5′-RACE procedure. Open boxes, putative −35 and −10 promoter elements as inferred from similarity to the E.coli σ70 consensus promoter. Dotted line, putative stem–loop structure that may be involved in transcription termination of 6S RNA transcripts, which would be in contradiction to a bioinformatic analysis predicting that hairpin structures at transcription termination sites may not be formed in A.aeolicus (37).
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fig5: 5′-RACE analysis of A.aeolicus 6S RNA transcripts. (A) Agarose gel analysis of 5′-RACE products using A.aeolicus total RNA and 6S RNA-specific primers for reverse transcription and PCR. M1, M2, DNA molecular weight markers, with the length (in bp) indicated on the left. −TAP, 5′-RACE without TAP treatment; +TAP, 5′-RACE including TAP digestion, additionally yielding product II which is inferred to represent a primary transcript with a 5′-triphosphate end. Controls: no RT, PCR of total RNA without a prior reverse transcription step; no template, PCR without template. (B) A.aeolicus DNA sequence of the 6S RNA coding region. Bold letters, reading frames pheT (β subunit of phenylalanyl-tRNA synthetase; stop codon marked by the gray box) and aq_1731 (5-formyltetrahydrofolate cyclo-ligase homolog; start codon indicated by gray oval). Small letters, putative 6S RNA coding region; for the 3′ boundary, see Figure 3. Nucleotides identified as 5′ ends of RNAs that yielded prominent bands in the RACE experiment are indicated by arrows with the roman number corresponding to the respective band on the gel in (A) (II, IV: each identified in 4 out of 4 sequenced clones; III, V: each arrow represented by 2 out of 4 sequenced clones); the 5′ end of product I was not determined. Horizontal solid arrow, position of oligonucleotide used as reverse transcription primer and northern probe (Figure 2C); horizontal broken arrow, 3′ primer for the final PCR step of the 5′-RACE procedure. Open boxes, putative −35 and −10 promoter elements as inferred from similarity to the E.coli σ70 consensus promoter. Dotted line, putative stem–loop structure that may be involved in transcription termination of 6S RNA transcripts, which would be in contradiction to a bioinformatic analysis predicting that hairpin structures at transcription termination sites may not be formed in A.aeolicus (37).

Mentions: The procedure for 5′-RACE was adapted from (16) and Christoph Jöchl (University of Innsbruck, Austria, PhD thesis). Briefly, 24 μg of total cellular RNA was incubated with 20 U DNase I (RNase-free, Roche) for 20 min at 37°C in a volume of 50 μl containing 10 mM sodium acetate (pH 5.2) and 0.5 mM MgCl2. After phenol/chloroform and chloroform extraction and ethanol precipitation, half the RNA was digested with 15 U tobacco acid pyrophosphatase (TAP, Eurogentec) in a total volume of 100 μl 1× TAP digestion buffer (50 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Triton X-100) in the presence of 20 U RNase Inhibitor (MBI Fermentas) for 40 min at 37°C; the other half was treated in the same manner, except that the TAP enzyme was omitted. Samples were again extracted with phenol/chloroform and chloroform, precipitated with ethanol and denatured for 5 min at 90°C in a volume of 24.5 μl double-distilled water (ddH2O) containing 1 nmol of adapter (5′-GTCAGCAATCCCTAACgag; capital letters denoting deoxyribonucleotides and lower case letters ribonucleotides). Ligation of the adapter to the denatured RNA was performed overnight at 4°C and another 3 h at room temperature in a final volume of 40 μl containing 0.01% BSA, 1× T4 RNA ligase buffer (MBI Fermentas), 1 mM ATP, 0.5 U/μl RNase inhibitor and 1.125 U/μl T4 RNA Ligase (MBI Fermentas). After phenol/chloroform and chloroform extractions and ethanol precipitation, nucleic acids were redissolved in 36 μl ddH2O. An aliquot of 4.5 μl was then denatured together with 0.5 μl (1 pmol) of the gene-specific primer 5′-CTGCCGCAGTGCAGGAAGTGCCGT (see Figure 5B, horizontal solid arrow) at 65°C for 5 min. The sample was immediately put on ice and supplemented with 5 μl of reverse transcription mix containing 2× reverse transcription buffer (Invitrogen; 100 mM Tris acetate pH 8.4, 150 mM potassium acetate, 16 mM magnesium acetate, stabilizers), 2 mM dNTPs, 0.01 M DTT, 1.5 U/μl Thermoscript Reverse Transcriptase (Invitrogen) and 2 U/μl RNase inhibitor, followed by incubation for 5 min at 42°C, 20 min at 55°C, 20 min at 60°C, 20 min at 65°C and 5 min at 85°C. RNA templates were then digested with 2.5 U RNase H (New England Biolabs) at 37°C for 20 min. After PCR amplification with the adapter-specific primer 5′-GTCAGCAATCCCTAACGAG and the gene-specific primer 5′-GAGCTTTAAGGTGGGAAGTC (Figure 5B, horizontal broken arrow), prominent bands were excised from a 2% agarose gel, eluted, re-amplified, again gel-purified and finally cloned utilizing the TOPO-cloning system (Invitrogen). For each prominent amplification product, several plasmid clones were sequenced.


Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog.

Willkomm DK, Minnerup J, Hüttenhofer A, Hartmann RK - Nucleic Acids Res. (2005)

5′-RACE analysis of A.aeolicus 6S RNA transcripts. (A) Agarose gel analysis of 5′-RACE products using A.aeolicus total RNA and 6S RNA-specific primers for reverse transcription and PCR. M1, M2, DNA molecular weight markers, with the length (in bp) indicated on the left. −TAP, 5′-RACE without TAP treatment; +TAP, 5′-RACE including TAP digestion, additionally yielding product II which is inferred to represent a primary transcript with a 5′-triphosphate end. Controls: no RT, PCR of total RNA without a prior reverse transcription step; no template, PCR without template. (B) A.aeolicus DNA sequence of the 6S RNA coding region. Bold letters, reading frames pheT (β subunit of phenylalanyl-tRNA synthetase; stop codon marked by the gray box) and aq_1731 (5-formyltetrahydrofolate cyclo-ligase homolog; start codon indicated by gray oval). Small letters, putative 6S RNA coding region; for the 3′ boundary, see Figure 3. Nucleotides identified as 5′ ends of RNAs that yielded prominent bands in the RACE experiment are indicated by arrows with the roman number corresponding to the respective band on the gel in (A) (II, IV: each identified in 4 out of 4 sequenced clones; III, V: each arrow represented by 2 out of 4 sequenced clones); the 5′ end of product I was not determined. Horizontal solid arrow, position of oligonucleotide used as reverse transcription primer and northern probe (Figure 2C); horizontal broken arrow, 3′ primer for the final PCR step of the 5′-RACE procedure. Open boxes, putative −35 and −10 promoter elements as inferred from similarity to the E.coli σ70 consensus promoter. Dotted line, putative stem–loop structure that may be involved in transcription termination of 6S RNA transcripts, which would be in contradiction to a bioinformatic analysis predicting that hairpin structures at transcription termination sites may not be formed in A.aeolicus (37).
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Related In: Results  -  Collection

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fig5: 5′-RACE analysis of A.aeolicus 6S RNA transcripts. (A) Agarose gel analysis of 5′-RACE products using A.aeolicus total RNA and 6S RNA-specific primers for reverse transcription and PCR. M1, M2, DNA molecular weight markers, with the length (in bp) indicated on the left. −TAP, 5′-RACE without TAP treatment; +TAP, 5′-RACE including TAP digestion, additionally yielding product II which is inferred to represent a primary transcript with a 5′-triphosphate end. Controls: no RT, PCR of total RNA without a prior reverse transcription step; no template, PCR without template. (B) A.aeolicus DNA sequence of the 6S RNA coding region. Bold letters, reading frames pheT (β subunit of phenylalanyl-tRNA synthetase; stop codon marked by the gray box) and aq_1731 (5-formyltetrahydrofolate cyclo-ligase homolog; start codon indicated by gray oval). Small letters, putative 6S RNA coding region; for the 3′ boundary, see Figure 3. Nucleotides identified as 5′ ends of RNAs that yielded prominent bands in the RACE experiment are indicated by arrows with the roman number corresponding to the respective band on the gel in (A) (II, IV: each identified in 4 out of 4 sequenced clones; III, V: each arrow represented by 2 out of 4 sequenced clones); the 5′ end of product I was not determined. Horizontal solid arrow, position of oligonucleotide used as reverse transcription primer and northern probe (Figure 2C); horizontal broken arrow, 3′ primer for the final PCR step of the 5′-RACE procedure. Open boxes, putative −35 and −10 promoter elements as inferred from similarity to the E.coli σ70 consensus promoter. Dotted line, putative stem–loop structure that may be involved in transcription termination of 6S RNA transcripts, which would be in contradiction to a bioinformatic analysis predicting that hairpin structures at transcription termination sites may not be formed in A.aeolicus (37).
Mentions: The procedure for 5′-RACE was adapted from (16) and Christoph Jöchl (University of Innsbruck, Austria, PhD thesis). Briefly, 24 μg of total cellular RNA was incubated with 20 U DNase I (RNase-free, Roche) for 20 min at 37°C in a volume of 50 μl containing 10 mM sodium acetate (pH 5.2) and 0.5 mM MgCl2. After phenol/chloroform and chloroform extraction and ethanol precipitation, half the RNA was digested with 15 U tobacco acid pyrophosphatase (TAP, Eurogentec) in a total volume of 100 μl 1× TAP digestion buffer (50 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Triton X-100) in the presence of 20 U RNase Inhibitor (MBI Fermentas) for 40 min at 37°C; the other half was treated in the same manner, except that the TAP enzyme was omitted. Samples were again extracted with phenol/chloroform and chloroform, precipitated with ethanol and denatured for 5 min at 90°C in a volume of 24.5 μl double-distilled water (ddH2O) containing 1 nmol of adapter (5′-GTCAGCAATCCCTAACgag; capital letters denoting deoxyribonucleotides and lower case letters ribonucleotides). Ligation of the adapter to the denatured RNA was performed overnight at 4°C and another 3 h at room temperature in a final volume of 40 μl containing 0.01% BSA, 1× T4 RNA ligase buffer (MBI Fermentas), 1 mM ATP, 0.5 U/μl RNase inhibitor and 1.125 U/μl T4 RNA Ligase (MBI Fermentas). After phenol/chloroform and chloroform extractions and ethanol precipitation, nucleic acids were redissolved in 36 μl ddH2O. An aliquot of 4.5 μl was then denatured together with 0.5 μl (1 pmol) of the gene-specific primer 5′-CTGCCGCAGTGCAGGAAGTGCCGT (see Figure 5B, horizontal solid arrow) at 65°C for 5 min. The sample was immediately put on ice and supplemented with 5 μl of reverse transcription mix containing 2× reverse transcription buffer (Invitrogen; 100 mM Tris acetate pH 8.4, 150 mM potassium acetate, 16 mM magnesium acetate, stabilizers), 2 mM dNTPs, 0.01 M DTT, 1.5 U/μl Thermoscript Reverse Transcriptase (Invitrogen) and 2 U/μl RNase inhibitor, followed by incubation for 5 min at 42°C, 20 min at 55°C, 20 min at 60°C, 20 min at 65°C and 5 min at 85°C. RNA templates were then digested with 2.5 U RNase H (New England Biolabs) at 37°C for 20 min. After PCR amplification with the adapter-specific primer 5′-GTCAGCAATCCCTAACGAG and the gene-specific primer 5′-GAGCTTTAAGGTGGGAAGTC (Figure 5B, horizontal broken arrow), prominent bands were excised from a 2% agarose gel, eluted, re-amplified, again gel-purified and finally cloned utilizing the TOPO-cloning system (Invitrogen). For each prominent amplification product, several plasmid clones were sequenced.

Bottom Line: The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus.We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches.By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

View Article: PubMed Central - PubMed

Affiliation: Philipps-Universität Marburg, Institut für Pharmazeutische Chemie Marbacher Weg 6, D-35037 Marburg, Germany.

ABSTRACT
By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its gamma-proteobacterial homologs (approximately 185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the gamma-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the gamma-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites.

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