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RNA – A New Role Amy Anderson
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Introduction – How’d they do that?
40+ years, still no complete answer… On You! Off You! Gene Gene Gene Gene How does gene expression get turned on and off?
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Anabaena Heterocysts How do the cells differentiate?
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So we need something new.
Originally researchers looked for proteins… …GCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGTTCACTCCGTGAGTAAGTTTTTGTAATTAACGT… Transcription …but nothing was found to account for everything. So we need something new.
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Where to look? E. coli!
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This small RNA is called rhyB.
E. coli’s Regulatory Mechanism – small RNA Small DNA sequence in between two genes Transcribed RNA bound to mRNA of targeted genes Binding blocked mRNA from being translated. Gene 1 Gene 2 …AATGCGCTA…GAATTATCGCG… 3. Blocks mRNA translation into protein This small RNA is called rhyB. It regulates the expression of proteins associated with the presence of iron. 1. Transcribed into RNA GCUA…GAAUUA 2. sRNA binds to mRNA containing targeted gene …GCUCUUACGUCGUCGAUUGCAGCGUA…
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Regulation of rhyB in E.coli
RNA Polymerase Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 ……..ACTAAAGTTGCCAATGTCAAAGATTTAGTAGAGACTTGCATATCGA…….TTCACTCCGTGAGTAAGTTTTTGTAATTAACGT…….. rhyB rhyB Promoter site Fe+2 fur Fe+2 Fe+2 Fe+2 Fe+2 Fe+2 Fe+2
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! Secondary Structure of rhyB mRNA of targeted gene rhyB binding site
GGAGAAC UAUUACUU UUU ! …. GUUAACUAUUTUTTGAUGUGCCAA…. rhyB binding site mRNA of targeted gene
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DNA of multiple targeted genes
mRNA of targeted genes with rhyB binding site
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Does Anabaena have a similar mechanism?
My Strategy: - BLAST the intergenic region surrounding each motif. - Look for partial matches that lie antisense (found on the opposite strand of) a gene (preferably one from my list). What I was given: - Set of motifs (sequences) that frequently showed up upstream from possible heterocyst related genes. - A list of possible heterocyst related genes What I was looking for: - Sequences that looked like an RNA (lollypops on a bubble) Motifs: M10 - CACGTTATCTGTTGAGACCGGGTGTAAGGGTTT M11 - TACACCCTTTTCCAAACCCTTGATCTTTCGTTTTCATGCGTAAGT M12 - AAAACTCTACCCACAAGGGGATAGAGTTTTGTCAGTGGTCAGTGG …… Gene 1 Gene 2 …AATGCGCTA…GAATTATCGCG… …AATGCGCTA…GAATTATCGCG… …TTACGCGAT…CTTAATAGCGC… …AATGCGCTA…GAATTATCGCG… …TTACGCGAT…CTTAATAGCGC…
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Intergenic regions Motif 10 was found in:
asl1111 alr1112 ACGT…M10…AGGT alr2744 alr2745 ACGT…M10…AGGT D5 P13 asl1274 asr1275 ACGT…M10…AGGT all2909 all2908 ACGT…M10…AGGT D7 P16 asl2850 alr2851 ACGT…M10…AGGT all3558 all3557 ACGT…M10…AGGT D15 P19 all3239 alr3240 ACGT…M10…AGGT alr3693 asr3694 ACGT…M10…AGGT D17 P20 all4668 asr4669 ACGT…M10…AGGT all4219 all4218 ACGT…M10…AGGT D24 P21 all1124 all1123 ACGT…M10…AGGT alr4468 alr4469 ACGT…M10…AGGT P6 P22 all1683 all1682 ACGT…M10…AGGT P10 alr4788 all4789 ACGT…M10…AGGT C26 all1782 all1781 ACGT…M10…AGGT P11
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BLAST hit alr0787 all0788 12-15 nucleotide overlap Most intergenic regions containing motif 10 hit this spot.
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Took an alignment of the portions that hit all0788.
Seq Seq GGGTGTA Seq Seq Seq Seq Seq GGGTGTA Seq Seq CAGGTTATCT GTTGAGACCG GGTGTAAGGG TTTAAGGGTA CAGGGGTGTA Seq AGGTTATCT GTTGAGACCG GGTGTAAGGG TTTAAGGGTA CAGGGGTGTA Seq GGGTGTA Seq GGGTGTA consensus 1 Seq ACCC CTAC ACCCTTCTC Seq AGGGTTTCAA AAATTTATAC CCCTATACCC CTAC ACCCTTGTC Seq ATACCC CTAC AACCTTTTC Seq ATACCC CTAC ACCCTTGTC Seq CCC CTAT ACCCTTGTC Seq ACCCTTGTC Seq AGGGTTTCAA GCATTTATAC CCTTATACCC CTAC ACCCTTGTC Seq ATACCC CCAT ACCCTTGTT Seq AGGGTTTCAA GCATTTATAC TCCTACATCC CTAC ATCCTTGTC Seq AGGGTTTCAA GCATTTATAC CCTTATACCC CTAC ACCCTTGTC Seq AGGGTTTCAA GCATTTATAC CCTTGTATCC CTATCCCATA CACCCTTATC Seq AGGGTTTCAA GCATTTATAC CCTCATACCC CTAT ACCCTTGTC consensus * **** * Seq CAAACCCTTG ATTTCTC-GT TTTCATGCGT AAGTCCTA- Seq TAAACCCTTG ATTTTTC-GT TTTCATGCGT AAGTCCTA- Seq CAAAACCTTG ATCTTTC-GT TTTCATGTGT AAGTCCTA- Seq CAAACCCTCG ATCTTTC-GT TTTCATGCGT AAGTCCTA- Seq CAAACCCTTG ATGTTTT-AT TTTTCTGCGT AAGTCCTAT Seq CAAACCCTTG ATCTTTC-GT TTTCATGCGT AAGTCCTA- Seq CAAACCCTTG ATCTTTCCGT TTTCATGCGT AAGTCCTA- Seq CAAACCCTTG ATCTTTC-GG TTTCATGCGT AAGTC---- Seq CAAACCCTTG ATCTTTC-GT TTTCATGCCT AAGTCCTA- Seq CAAACCCTCG ATCTTTC-GT TTTCATGCGT AAGT----- Seq CAAAGCCTTG ATATTTC-TT TTTCATGGGT AAGTC---- Seq CAAACCCTTT ATCTTTC-GT TTTCATGCGT AAGTC---- consensus *** *** ** * * *** ** * ****
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Alignment of sub-sequences
Seq GGGTGTAAGG GTTTCAAGCA TTTATACCCT TGTATCCCTA TCCCATACAC Seq GGGTGTAAGG GTTTCAAGCA TTTATACCCT CATACCCCTA T AC Seq GGGTGTAAGG GTTTCAAGCA TTTATACCCT TATACCCCTA C AC Seq GGGTGTAAGG GTTTCAAGCA TTTATACCCT TATACCCCTA C AC Seq GGGTGTAAGG GTTTCAAAAA TTTATACCCC TATACCCCTA C AC Seq GGGTGTAAGG GTTTCAAACA TTTATACCCC TAAACCCCTA T AT Seq GGGTGTAAGG GTTTCAAGCA TTTATACTCC TACATCCCTA C AT consensus ********** ******* * ******* * * ***** * Seq CCTTATCCAA AGCCTTGATA TTTCT-TTTT CATGGGTAAG TC---- Seq CCTTGTCCAA ACCCTTTATC TTTCG-TTTT CATGCGTAAG TC---- Seq CCTTGTCCAA ACCCTCGATC TTTCG-TTTT CATGCGTAAG T----- Seq CCTTGTCCAA ACCCTTGATC TTTCCGTTTT CATGCGTAAG TCCTA- Seq CCTTGTCTAA ACCCTTGATT TTTCG-TTTT CATGCGTAAG TCCTA- Seq CCTTCTCCAA ACCCTTAATC TTTAG-TTTT CATACGTAAG TCCTAT Seq CCTTGTCCAA ACCCTTGATC TTTCG-TTTT CATGCCTAAG TCCTA- consensus **** ** ** * *** ** *** **** *** **** *
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Secondary structures of aligned sequences
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BLASTed this bubble sequence
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Some BLAST Results patN About 80% landed in between parallel genes
alr2966 alr2967 alr2968 all4019 all4020 all4021 all4022 all4023 alr4810 alr4811 alr4812 patN
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Summary and Conclusions
More work needs to be done. It is possible that Anabaena employes small RNA as a regulatory mechanism (I found nothing that suggested otherwise). When looking for any kind of pattern or sequence you must always consider the probability that it would happen by chance. The data I showed was just a fraction of everything I had gathered. I didn’t even scratch the surface of this new topic, but I learned a lot trying.
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Thanks to: Jeff Elhai Peter Wolk For being so patient with me :^)
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