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Characterization of the SMK box: A riboswitch that binds S-adenosylmethionine

Fuchs, Ryan T.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1251830925

Abstract Details

2009, Doctor of Philosophy, Ohio State University, Microbiology.
Riboswitches are RNA sequences that regulate downstream gene expression via changes in RNA structure. Riboswitches respond to a stimulus, typically binding of an effector molecule, that causes a structural rearrangement without additional trans-acting factors. The rearrangement can cause or prevent formation of an intrinsic transcriptional terminator to regulate at the level of transcription termination, or it can modulate the accessibility of the Shine-Dalgarno (SD) region to regulate at the level of translation initiation. There are a couple classes of riboswitches that bind S-adenosylmethionine (SAM). The S box riboswitches are found in a wide range of bacteria while the SAM-II riboswitch is found primarily in Proteobacteria. Both riboswitches recognize SAM in a similar manner, but they are very different in terms of sequence, secondary and tertiary structure. We discovered a third SAM binding riboswitch (called the SMK box) upstream of metK (encoding SAM synthetase) in many Lactobacillales species. SMK box RNA was shown to bind SAM and discriminate against S-adenosylhomocysteine. Through enzymatic probing, it was determined that SAM causes rearrangement in the RNA which results in sequestration of the SD region. This observation was confirmed by X-ray crystallography, which also showed that part of the SD contacts SAM. A translational fusion of the Enterococcus faecalis metK leader to lacZ was introduced into Bacillus subtilis and when the cells were grown under conditions in which SAM pools are elevated, β-galactosidase activity decreased. In contrast, a transcriptional fusion showed no effect. This suggests that the SMK box riboswitch down-regulates gene expression at the level of translation in response to SAM. This was supported by the discovery that SAM inhibits binding of 30S subunits to SMK box RNA. Mutant SMK box riboswitches that are deficient in SAM binding showed no SAM-dependent effect on 30S subunit binding and no regulation of lacZ expression. Quantitative PCR was utilized in order to analyze the metK transcript in vivo in a native organism, E. faecalis. The abundance of metK transcript in E. faecalis was found to be constant during growth under conditions resulting in high or low SAM. This supports the model that regulation of metK is not at the level of transcription. The half-life (t1/2) of the metK transcript was determined to be ~3 min under all conditions. In contrast, the t1/2 of the interaction between SAM and the SMK box in vitro is 7.8 sec (A. Smith). Thus, the interaction of the SMK box with its effector was shown to be of much shorter duration than the persistence of the transcript in the cell, suggesting that the regulatory effect and structural changes resulting from SAM binding are reversible. Attempts were made to demonstrate SMK box-mediated regulation in E. faecalis by directly measuring SAM synthetase activity and by constructing a metK-gusA fusion. Neither showed regulation of expression in response to changing SAM pools. The basis for these observations is unknown. It is possible that other factors are involved in regulation of metK in E. faecalis and they are masking the effect of the SMK box in vivo.
Tina Henkin, PhD (Advisor)
Charles Daniels, PhD (Committee Member)
Juan Alfonzo, PhD (Committee Member)
Kurt Fredrick, PhD (Committee Member)
139 p.
Microbiology
Riboswitch; SAM

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Citations

  • Fuchs, R. T. (2009). Characterization of the SMK box: A riboswitch that binds S-adenosylmethionine [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1251830925

    APA Style (7th edition)

  • Fuchs, Ryan. Characterization of the SMK box: A riboswitch that binds S-adenosylmethionine. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1251830925.

    MLA Style (8th edition)

  • Fuchs, Ryan. "Characterization of the SMK box: A riboswitch that binds S-adenosylmethionine." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1251830925

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.