Advertisement

Preparation and Crystallization of Riboswitches

  • Alla Peselis
  • Ang Gao
  • Alexander Serganov
Part of the Methods in Molecular Biology book series (MIMB, volume 1320)

Abstract

Recent studies have revealed that the majority of biological processes are controlled by noncoding RNAs. Among many classes of noncoding RNAs, metabolite-sensing segments of mRNAs called riboswitches are unique. Discovered over a decade ago in all three kingdoms of life, riboswitches specifically and directly interact with various metabolites and regulate expression of multiple genes, often associated with metabolism and transport of small molecules. Thus, riboswitches do not depend on proteins for binding to small molecules and play a role as both metabolite sensors and effectors of gene control. Riboswitches are typically located in the untranslated regions of mRNAs where they form alternative structures in the presence and absence of the ligand and modulate expression of genes through the formation of regulatory elements. To understand the mechanism of the riboswitch-driven gene control, it is important to elucidate how riboswitches interact with cognate and discriminate against non-cognate ligands. Here we outline the methodology to synthesize riboswitch RNAs and prepare riboswitch–ligand complexes for crystallographic and biochemical studies. The chapter describes how to design, prepare, and conduct crystallization screening of riboswitch–ligand complexes. The methodology was refined on crystallographic studies of several riboswitches and can be employed for other types of RNA molecules.

Key words

B12 riboswitch Fluoride riboswitch RNA secondary structure Crystallization 

References

  1. 1.
    Mironov AS, Gusarov I, Rafikov R et al (2002) Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111:747–756PubMedCrossRefGoogle Scholar
  2. 2.
    Nahvi A, Sudarsan N, Ebert MS et al (2002) Genetic control by a metabolite binding mRNA. Chem Biol 9:1043PubMedCrossRefGoogle Scholar
  3. 3.
    Winkler W, Nahvi A, Breaker RR (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419:952–956PubMedCrossRefGoogle Scholar
  4. 4.
    Sudarsan N, Barrick JE, Breaker RR (2003) Metabolite-binding RNA domains are present in the genes of eukaryotes. RNA 9:644–647PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Breaker RR (2011) Prospects for riboswitch discovery and analysis. Mol Cell 43:867–879PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Nudler E, Mironov AS (2004) The riboswitch control of bacterial metabolism. Trends Biochem Sci 29:11–17PubMedCrossRefGoogle Scholar
  7. 7.
    Winkler WC, Breaker RR (2005) Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol 59:487–517PubMedCrossRefGoogle Scholar
  8. 8.
    Serganov A, Nudler E (2013) A decade of riboswitches. Cell 152:17–24PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Serganov A, Patel DJ (2012) Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Annu Rev Biophys 41:343–370PubMedCrossRefGoogle Scholar
  10. 10.
    Milligan JF, Groebe DR, Witherell GW et al (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Pikovskaya O, Serganov AA, Polonskaia A et al (2009) Preparation and crystallization of riboswitch-ligand complexes. Methods Mol Biol 540:115–128PubMedCrossRefGoogle Scholar
  12. 12.
    Peselis A, Serganov A (2012) Structural insights into ligand binding and gene expression control by an adenosylcobalamin riboswitch. Nat Struct Mol Biol 19:1182–1184PubMedCrossRefGoogle Scholar
  13. 13.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Martin CT, Muller DK, Coleman JE (1988) Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry 27:3966–3974PubMedCrossRefGoogle Scholar
  15. 15.
    Rong M, Durbin RK, McAllister WT (1998) Template strand switching by T7 RNA polymerase. J Biol Chem 273:10253–10260PubMedCrossRefGoogle Scholar
  16. 16.
    Pleiss JA, Derrick ML, Uhlenbeck OC (1998) T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates. RNA 4:1313–1317PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Helm M, Brule H, Giege R et al (1999) More mistakes by T7 RNA polymerase at the 5′ ends of in vitro-transcribed RNAs. RNA 5:618–621PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Nacheva GA, Berzal-Herranz A (2003) Preventing nondesired RNA-primed RNA extension catalyzed by T7 RNA polymerase. Eur J Biochem 270:1458–1465PubMedCrossRefGoogle Scholar
  19. 19.
    Price SR, Ito N, Oubridge C et al (1995) Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J Mol Biol 249:398–408PubMedCrossRefGoogle Scholar
  20. 20.
    Martick M, Scott WG (2006) Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126:309–320PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Ferre-D'Amare AR, Zhou K, Doudna JA (1998) Crystal structure of a hepatitis delta virus ribozyme. Nature 395:567–574PubMedCrossRefGoogle Scholar
  22. 22.
    Serganov A, Rak A, Garber M et al (1997) Ribosomal protein S15 from Thermus thermophilus-cloning, sequencing, overexpression of the gene and RNA-binding properties of the protein. Eur J Biochem 246:291–300PubMedCrossRefGoogle Scholar
  23. 23.
    Xiong AS, Yao QH, Peng RH et al (2006) PCR-based accurate synthesis of long DNA sequences. Nat Protoc 1:791–797PubMedCrossRefGoogle Scholar
  24. 24.
    Walker SC, Avis JM, Conn GL (2003) General plasmids for producing RNA in vitro transcripts with homogeneous ends. Nucleic Acids Res 31:e82PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Studier FW, Rosenberg AH, Dunn JJ et al (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular PharmacologyNew York University School of MedicineNew YorkUSA
  2. 2.Department of Biochemistry and Molecular PharmacologyNew York University School of MedicineNew YorkUSA

Personalised recommendations