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Prokaryotic Regulatory RNAs Cole Franks Proteins have been known to have regulatory actions for many years. Prokaryotic regulatory proteins are particularly well understood; allosteric enzymes have been known since the 1960’s to carry out negative feedback. It seems, however, that proteins are far from the whole regulatory story. Evidence has been compiling for regulation by RNA itself. Most are familiar with the idea that DNA is transcribed to a single strand of mRNA, which is then translated to proteins by ribosomes. RNA structures such as small RNA, riboswitches, T boxes, and translation mediated transcription attenuation mechanisms have recently been shown to have an undeniable contribution to regulation of the prokaryotic genome. They also have some analogs in the eukaryotic genome. These RNA structures suggest that researchers have been overlooking a great deal of the picture when seeking to understand regulation, since they behave quite differently than most regulatory proteins. They function post-transcriptionally or by attenuating transcription, and are often attached to the mRNAs they regulate. They also provide further support for the RNA world hypothesis - the idea that RNA carried out more complex functions in organisms before protein evolved. This paper will provide an overview of some of these RNA structures in prokaryotes. The impact of post-transcriptional regulation is much more widespread than originally supposed. Nearly fifty-four percent of the proteins in the Salmonella proteome are indirectly or directly affected by small RNA regulation (Ansong et al. 2009). The aforementioned study was conducted by proteomic analysis after knocking out (disabling) the Hfq protein. Hfq is a protein known as an RNA chaperone, and is crucial for the function of some small RNAs. Most small RNAs (sRNAs) function by base pairing with mRNAs, causing the mRNAs to be degraded and/or preventing their translation. Hfq is so crucial because it binds single stranded RNA that is rich in AU (adjacent adenine and uracil nucleotides); it therefore binds with sRNA and aids in its intracellular stability and its binding to target mRNA (reviewed in Vogel, 2009). Hfq, a hexameric ring protein, works in several ways. It remodels sRNAs to remove structures which would inhibit binding with mRNA, modulates sRNA levels, protects sRNAs from degradation until they manage to base pair with an mRNA, and may even serve to recruit RNA degradation machinery to break down sRNA-mRNA complexes (reviewed in Waters and Storz, 2009). The hfq knockout shows the sheer importance of RNA regulation. The mechanisms of sRNA action are quite variable. Some even bind proteins rather than mRNA. CsrA is an RNA binding protein that binds to mRNA to regulate the translation of proteins involved in carbon usage. Two sRNAs, CsrB and CsrC, bind this protein to prevent it from contacting the 5’ untranslated region of the mRNA so it cannot affect translation (reviewed in Babitzke and Romeo, 2007). Most sRNAs, however, bind a complementary or partly complementary mRNA. There are two major varieties of such base pairing sRNA. Cis encoded antisense sRNA (encoded from the same region of DNA) is often encoded from the complementary DNA strand opposite the target RNA, and has long stretches of complementary base pairs (reviewed in Brantl, 2007). Cis-encoded sRNA does not usually need hfq to help it anneal with the target mRNA; it anneals more easily because of how thoroughly complementary it is to the target. Though the sRNA and its target mRNA are encoded from the same stretch of DNA, they act as two separate molecules in the cell. In plasmids and transposons, they function to maintain the appropriate number of copies of the mobile element. The cis-encoded sRNAs use several mechanisms to prevent the mobile element, which is a piece of DNA that has been transferred from another part of the organism’s chromosome or from another organism, from being copied too many times. Two of these mechanisms are the inhibition of replication primer formation and transposase translation. Primers are short sequences required to replicate regions of DNA, and transposase “cut and pastes” transposons to other parts of the genome. Cis encoded sRNAs from the other parts of the bacterial chromosome are not well understood. Some of these regulate proteins that are necessary, but are toxic in high amounts. Others affect the expression of genes involved in operons (reviewed in Waters and Storz, 2009) Trans encoded sRNA action is better understood than that of cis encoded sRNA. These sRNA have only limited complementarity, and are functionally analogous to miRNA in eukaryotes. Trans encoded sRNAs often bind to the 5’ untranslated region (the region of the mRNA upstream from the coding region) and cover up the ribosome binding site, which is a sequence of base pairs known as the Shine-Dalgarno sequence. After they bind to the mRNA, RNase-E degrades the sRNA-mRNA complex. Another mechanism is activation of translation by an antiantisense mechanism, in which the binding of the sRNA to the mRNA disrupts a secondary inhibitory structure on the mRNA and allows it to be expressed. Because these have limited complementarity, they only require a small core of base pairings to function, and can have multiple interactions. While usually these sRNAs require Hfq to anneal, they may also function when there are high concentrations of the sRNA. Since these sRNA are trans encoded, there is little correlation between their location in the genome and the location of their target gene. They are often synthesized by prokaryotes under very strict growth conditions (reviewed in Waters and Storz, 2009). Since mainly trans encoded sRNAs requires Hfq, it is likely that in knockout studies on hfq, the main type of sRNA tested is trans-encoded sRNA. Salmonella is a well understood gram negative bacteria which has recently been studied as a model organism for RNA mediated regulation. When hfq was knocked out in Salmonella under varying growth conditions, the bacteria had a decreased growth rate, had decreased virulence, and decreased ability to replicate inside macrophages, a type of white blood cell (reviewed in Ansong et al. 2009). Hfq knockout altered expression in about fifty percent of Salmonella’s proteins when tested across several media conditions. The impacts were widespread; Hfq was crucial in dealing with stressful growth conditions, multiple kinds of metabolism, lipopolysaccharide synthesis (essential for virulence in Gram negative bacteria), carbon usage, and other processes. Since transcripts of mRNA for all tested proteins remained the same in hfq knockouts, trans-encoded sRNAs must have a huge impact on posttranscriptional regulation (Ansong et al. 2009). There are several other noteworthy mechanisms of RNA post-transcriptional and transcription attenuation regulation. One was discovered in examinations of the Btub and Cob operons in E. Coli and Salmonella, respectively. These operons are involved in the synthesis of B12 coenzyme, which is needed to use vitamin B12. It was previously known that these operons have feedback relationships with coenzyme B12, and that the 5’ untranslated regions of their mRNAs were required for this feedback to work. However, no protein was implicated in interactions with coenzyme B12 and the mRNA. It was also noteworthy that the 5’ untranslated regions of the mRNAs from the operons reorganized themselves by spontaneous cleavage in the presence of coenzyme B12. Analysis showed that there was actually direct binding of coenzyme B12 by these mRNAs (Mandal and Breaker, 2004). This mechanism is known as a riboswitch – a structure in the 5’ untranslated region of mRNA that detects the metabolite coded for by the mRNA and alters the structure of the 5’ untranslated region to affect translation or transcription of the RNA. Riboswitches typically have two domains (Figure 1). The aptamer domain recognizes the target molecule, and the expression platform reorganizes the 5’ untranslated region of the mRNA in response to the binding of the metabolite. There are two main mechanisms used by the expression platform. The first regulates RNA transcription by forming an intrinsic terminator stem. An intrinsic terminator is a stem loop structure that is usually followed by six or more U residues. This structure causes RNA polymerase to abort transcription before the coding portion has been made. Put simply, the aptamer domain is transcribed, and if the metabolite is present the riboswitch binds the molecule and rearranges its structure to prevent the coding region of the mRNA from being transcribed. When there are low concentrations of metabolite, the unbound aptamer domain allows the formation of an antiterminator stem. The formation of this stem prevents the terminator stem from forming, and allows the mRNA to be fully transcribed. The other mechanism operates by altering translation initiation. When the metabolite binds to the aptamer domain, the expression platform alters the 5’ untranslated region to keep the ribosome from accessing the ribosome binding site and prevents translation. There is also a possibility that if the terminator stem were formed by base pairing with the ribosome binding site, transcription and translation could be controlled simultaneously – the terminator stem would stop any more mRNA from being transcribed, and any that had passed the point of transcription termination could not be translated (Mandal and Breaker, 2004) These mechanisms are common to the coenzyme B12 riboswitch, but evidence exists that other riboswitches share these mechanisms (reviewed in Mandal and Breaker, 2004). Riboswitches appear to have diverse structures and mechanisms; in some cases they may even act as ON switches instead of OFF switches (Mandal and Breaker, 2004). Two other mechanisms of transcription-attenuation regulation by prokaryotes are translation mediated transcription attenuation mechanisms and T- boxes. Translation mediated transcription attenuation mechanisms use the speed of translation, which is determined by the concentration of aminoacylated tRNA, to prevent or facilitate the formation of a terminator stem. They usually affect amino acid operons because in the leader regions of the operon being regulated there are several adjacent codons that code for the pertinent amino acid (Figure 2). If the ribosome races down these codons, indicating a high concentration of aminoacylated tRNA, the terminator forms in the leader transcript. If the ribosome stalls, the nascent mRNA that was transcribed during the stalling folds to form an antiterminator stem, which prevents the formation of a terminator stem. This is the mechanism used by many Gram negative prokaryotes, with some variation. In the tryptophan operon in E. Coli, there is also a pause hairpin. This pause hairpin is crucial – it allows the stalled ribosome to catch up with transcription. The pause hairpin, like the antiterminator, prevents the terminator stem from forming. It forms while the ribosome stalls and prevents further transcription (reviewed in Henkin and Yanofsky, 2002). T-boxes are hairpin structures in the 5’ untranslated region of mRNA (Figure 3). They regulate amino acid operons in Gram positive bacteria. This structure has a specifier hairpin, which has an anti-anti codon. This is a codon complementary to the anticodon in the tRNA of the amino acid for the particular operon. This anti-anti codon binds to the tRNA, which in turn causes the mRNA to form an antiterminator stem by interaction of a 5’UGGN3’ sequence in the 5’ untranslated region and the complementary sequence on the 3’ end of the tRNA (Putzer et al. 1995, Yousef et al. 2005). Since the tRNA is unbound, this occurs when there are low concentrations of the required amino acid. The binding of the tRNA can also allow an antisequester of the ribosome binding site to form. An antisequester is a structure that forms to prevent another structure (a sequester) from folding the ribosome binding sequence in a fashion that would be inaccessible to the ribosome. Put in simple geometric terms, without tRNA binding two hairpin structures would already form in the T-box upon transcription. When the tRNA binds to the specifier hairpin, however, it “reaches” over and forms either an antiterminator or an antisequester that prevents a terminator or a sequester from forming (Vitreschak et al. 2008). These relatively newfound RNA structures show that RNA regulation deserves at least as much, if not more, attention than protein regulation. Not only can RNA attach to transcribed mRNA to cause degradation and prevent transcription, but it can also form built-in structures in the leading ends of transcripts to halt transcription and/or translation. These structures are also clearly important pieces of evidence in the argument for RNA world. Complex folded RNA structures suggest that at some point in time RNA could have had many more functions. How has non-coding RNA changed over time? What other functions can RNA have? What are the full impacts of these functions? Small RNA, riboswitches, Tboxes, and translation mediated transcription attenuation mechanisms raise many such questions. While many researchers continue to provide answers, what we still do not understand about RNA may be crucial to our understanding of life. Figure 1 (Mandal and Breaker, 2004) A) This is the transcription attenuation mechanism of a riboswitch. The terminators and antiterminator are in red, and one can see how the antiterminator binds with one of the stretches of terminator sequence when nothing binds with the aptamer domain. This prevents the terminator stem from forming, hereby maintaining transcription. B) This is the mechanism in which the ribosome binding site, or ShineDalgarno sequence, is sequestered in high concentrations of the metabolite. The RBS is in green, and the sequester and antisequester are in red. Figure 2 (Henkin and Yanofsky, 2002) Termination: High concentration of aminoacylated tRNA allows normal translation to occur, so a terminator forms. Antitermination: The ribosome stalls because of the low aminoacylated tRNA concentration while transcription continues. The nascent mRNA folds to form an antiterminator. Figure 3 (Vitreschak et al. 2008) A) In A and B, variants of the transcription attenuation mechanism of a T-box are shown. The uncharged tRNA allows an antiterminator to form, preventing termination of transcription. B) In C, the uncharged tRNA binds with the specifier hairpin and forms an antisequester, allowing translation. References: Mandal, M., & Breaker, R.R. (2004). Gene regulation by riboswitches. Nature Reviews:Molecular Cell Biology , 5. Retrieved from http://rnaworld.bio.ku.edu/reprints/main/Mandal(2004c)Nature_ Rev_Molec_Cell_Biol _5,451.pdf V itreschak, A.G. et al. (2008). Comparative genomic analysis of t-box regulatory systems in bacteria. RNA, 14. Retrieved from http://rnajournal.cshlp.org/content/14/4/717.full Henkin, T. M., & Yanofsky, C. (2002). Regulation by transcription attenuation in bacteria : how rna provides instructions for transcription termination/antitermination decisions. BioEssays, 24(8), Retrieved from http://rnaworld.bio.ku.edu/reprints/incoming/_misc/+Henkin()B ioEssays_24,700.%5BYanofsky,attenuation%5Dpdf.pdf Ansong, C. et al. (2009). Global systems -level analysis of hfq and smpb deletion mutants in salmonella : implications for virulence and global protein translation. Plos one, Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19277208 Vogel, J. (2009). A rough guide to the non-coding rna world of salmonella. Molecular Microbiology, Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19277208 Waters, L. S., & Storz, G. (2009). Regulatory rnas in bacteria. Cell, 136(4), Retrieved from http://www.cell.com/abstract/S0092 8674(09)00125-1 Babitzke, P., and Romeo, T. (2007). CsrB sRNA family: Sequestration of RNAbinding regulatory proteins. Curr. Opin. Microbiol. 10, 156–163. Brantl, S. (2007). Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr. Opin. Microbiol. 10, 102–109. Yousef, M.R., Grundy, F.J., and Henkin, T.M. 2005. Structural transitions induced by the interaction between tRNAGly and the Bacillus subtilis glyQS T box leader RNA. J. Mol. Biol. 349: 273– 287. Putzer, H., Laalami, S., Brakhage, A.A., Condon, C., and GrunbergManago, M. 1995. Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis: Induction, repression and growth-rate regulation. Mol. Microbiol. 16: 709–718.