COMMENTARY

+ Mg2 -dependent translational speed bump acts to regulate gene COMMENTARY Kelly T. Hughesa,1

DNA Transcription in (7). Normally, an mRNA in bacteria is described as The central dogma of molecular biology states that having a 5′UTR between the transcriptional start-site DNA is transcribed (by RNA polymerase) to messenger and the initiation codon. The 5′UTR is often RNA that is then translated (by ) into protein used in translational control by controlling the mRNA (1). The regulation of gene expression can occur at any secondary structure to either expose or occlude the ri- level: transcription, mRNA stability, translation that in- bosome binding site upstream of the translation initia- cludes mRNA folding to reveal or occlude translation tion codon. sequences upstream of the initiation sites, and protein modification and stability. In structural genes of many bacterial systems are eukaryotes, the processes of transcription and trans- used to effect transcriptional control, such as in the lation are carried out in the cell nucleus and cytoplasm, case of riboswitches. Riboswitches allow metabolites to respectively. In bacteria, which lack a nucleus, the pro- control alternative mRNA secondary structures in the cesses of transcription and translation are coupled. This 5′UTR region to facilitate either the formation or de- provides an evolutionary target that is unique to bacteria stabilization of a transcriptional terminator (8, 9). for controlling gene expression. Many gene regulatory mechanisms in bacteria use transcription–translation Control of DNA Transcription by Charged tRNA coupling to affect whether transcription elongation The discovery of a role for charged tRNA species in through specific single or multigene continues transcriptional control of the histidine biosynthetic or terminates in response to changing concentrations of operons led to the discovery that translation played the metabolic product associated with a given operon. a critical role in the control of transcription in many In bacteria, termination of transcription occurs when amino acid biosynthetic operons (10). DNA sequencing RNA polymerase is preceded by a stretch of RNA that is of amino acid biosynthetic operons revealed the pres- not being translated, which occurs in the 3′-untranslated ence of 5′-translated, short, leader peptide regions RNA following the final translation termination codon of preceding the structural genes of these operons. These a given operon. Transcription termination in bacteria translated leader peptide regions were an integral occurs by one of two mechanisms that is either de- component of mechanisms that allowed for regulation pendent on the Rho transcriptional terminal factor or by of transcription of amino acid biosynthetic operons in the Rho-independent mechanism that involves the for- response to end product amino acid levels (7). The mation of a folded stem-loop structure in the RNA “true” end product sensed in the regulation of these followed by a poly-U sequence (2–4). Rho protein rec- amino acid biosynthetic operons was not a given amino ognizes untranslated RNA that is rich in cytosine base acid itself. Rather, the regulatory mechanisms sensed residues (5). levels of a specific amino acid in its charged form as an Suppression of transcription termination in gene aminoacyl tRNA. The leader peptide includes codons regulation was first characterized as a mechanism to for the specific amino acid end-product of that amino control the timing of gene expression during the infection acid biosynthetic operon. Between the leader peptide growth cycle of bacteriophage λ in (6). coding sequence and the initiation codon of the first Here, antiterminator proteins, such as the λ N protein, gene in the biosynthetic operon resides a transcrip- must be transcribed, translated, and reach sufficient lev- tional attenuator sequence. Whether transcription els to bind termination sequences to prevent termination continues into the biosynthetic genes or attenuates at and allow transcription to continue into downstream the attenuator sequence depends on how the RNA genes that are needed later in the infection cycle. sequence that includes the attenuator folds. Alternative Suppression of transcription termination during RNA folding, that either promotes formation or de- transcription–translation coupling was later found to stabilization of the attenuator depends on the rate of regulate expression of amino acid biosynthetic operons translation by the through the leader peptide.

aDepartment of Biology, University of Utah, Salt Lake City, UT 84112 Author contributions: K.T.H. wrote the paper. The author declares no conflict of interest. See companion article on page 15096. 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1618222114 PNAS | December 27, 2016 | vol. 113 | no. 52 | 14881–14883 Downloaded by guest on September 28, 2021 + Because transcription and translation are coupled in bacteria, translation speedometer to sense Mg2 concentrations by Salmonella the initial ribosome that translates the leader peptide follows di- cells (17). The unique chemical interactions between magnesium + rectly behind the transcribing RNA polymerase complex (11). and phosphate make Mg2 essential in many cellular processes + When the ribosome reaches the leader peptide termination codon, involving nucleic acids. Mg2 is required for hundreds of enzymatic the untranslated sequence between the leader peptide stop codon reactions. Salmonella possesses three uptake systems for magne- + and the initiation codon of the first biosynthetic gene folds in a way sium ion. As an enteric pathogen, Salmonella uses low Mg2 con- that forms a transcriptional attenuator. When the levels of the amino centrations as one of multiple sensors in determining that it is inside acid end product of a specific amino acid biosynthetic pathway a host cell to control expression of genes required for virulence (18). drop, such that the codons in the leader peptide that code for that Magnesium and phosphate are also connected in controlling ex- amino acid are translated at a reduced rate, the unbound RNA se- pression of Salmonella virulence genes. A two-component regulatory quence between first translating ribosome and the transcribing RNA system, PhoQ-PhoP, originally described as a regulator of the phoN complex is extended to include bases in the 3′-end of the leader phosphatase gene, acts as an environmental sensor that effects peptide. Exposed 3′ leader peptide-coding bases are free to in- expression of 5% of the Salmonella genome, and is a primary teract with downstream RNA attenuator sequences, destabilize the regulator of genes required for Salmonella pathogenesis includ- + + attenuator form, and RNA polymerase continues to transcribe into ing Mg2 uptake systems (19). One Mg2 uptake system, mgtA,is the structural genes of the biosynthetic operon for that amino acid. This mechanism allows for the transcription of a given amino acid biosynthetic operon to depend on the levels of the specific amino In PNAS, Gall et al. demonstrate a novel, leader acyl tRNA for that operon and respond accordingly. peptide-dependent attenuation mechanism + One of the best-characterized attenuation control mechanisms that acts as a Mg2 -dependent translation + in bacteria for amino acid biosynthesis is that of the histidine speedometer to sense Mg2 concentrations by biosynthetic operon (his)ofSalmonella (12). The Salmonella his Salmonella operon includes nine structural genes. The first structural gene in cells. the his operon is preceded by a classic Rho-independent termi- nator, which is preceded by the his leader peptide sequence. The induced more than 100-fold in response to low magnesium in a his leader peptide codes for 16 amino acids, including 7 con- PhoQP-dependent manner (20, 21). secutive histidine codons (13). At high levels of charged histidyl aminoacyl tRNA, the first translating ribosome proceeds behind Slow Pro Translation as a Metabolite Sensor Mechanism RNA polymerase to the leader peptide termination codon, the In addition to PhoQP-dependent transcription of mgtA,Galletal. attenuator forms, and RNA polymerase comes off the DNA as now demonstrate a second control mechanism is involved that uses + transcription is terminated. At low histidyl-tRNA levels, the first aMg2 -dependent translational speedometer through a leader translating ribosome stalls in the stretch on consecutive histidine peptide sequence reminiscent of the attenuation control mecha- codons within the his leader peptide sequence and exposed RNA nisms in control of amino acid biosynthetic operon expression (17). + bases at the 3′-end of the his leader peptide interfere with the The Mg2 -dependent translation speedometer mechanism takes folding of the RNA into the attenuator form. advantage of the relatively slow translation of proline codons. The The properties of the his leader peptide in the his operon at- mgtA leader peptide contains a stretch of seven codons with four tenuation system were used to develop a translational speedometer proline codons spaced by intervening codons: Pro-Glu-Pro-Thr- that measures how fast ribosomes translate the codons in the his Pro-Leu-Pro. Anything that slows translation through the al- leader region that normally include the seven consecutive histidine ternating Pro codon region of the mgtA leader peptide results in codons (14). Substitution at position His5 in the his leader peptide de-attenuation of mgtA transcription. The mgtA attenuation system sequence for other codons was used to determine the relative evolved to allow reduced activity of translation factors in response + + speed that ribosomes translate individual codons in the context of to low Mg2 concentrations. The Mg2 -dependent translation the leader peptide sequence. A slower speed of translation through speedometer mechanism in mgtA wassolvedbyclassicgenetic a codon at the His5 increased the probability that the attenuator selection and screening methods. Mutants that resulted in de- + would not form. In contrast, a faster translation speed through the attenuationinhighMg2 conditions included conversion of in- His5 position in the leader peptide resulted in an increased proba- tervening codons to Pro codons, resulting in three consecutive bility of attenuator formation. The mechanism of sensing translation Pro residues and ribosome stalling. In addition, mutants de- speed by this attenuation mechanism proved sensitive to the de- fectiveinribosomal-associatedproteinsL27andL31involvedin gree that one could measure differences in speed of translation translation efficiency, as well as translation-defective mutants through synonymous codons by the same charged tRNA species. specific to Pro codon translation in trmD and efp. Whether or not + This study also demonstrated that translation of proline codons is Mg2 concentrations have an allosteric effect of the activities of slow, relative to the other amino acids, presumably because of the one of these translation factors remains to be determined. What nature of peptide bond formation with the unique proline amino is remarkable to think about is that, similar to riboswitches that acid. In addition, the peptidyl transfer step after GTP hydrolysis on can evolve to bind ligands to regulate transcription, the cell elongation factor (EF)-Tu was demonstrated to be slower, in vitro, for should be able to evolve the translational speedometer mech- “ ” prolinethanfor normal amino acids residues (15). Furthermore, in anism to effect gene regulation for any system to allow regula- the his attenuation system two successive proline residues were tion in response to any ion, metabolite, protein, and so forth that translated at the same relative speed as stop codons (14). Finally, a could have allosteric effects on the translation machinery. new translation factor, EF-P was recently discovered that allowed for more efficient translation through poly-proline codons (16). Acknowledgments In PNAS, Gall et al. demonstrate a novel, leader peptide- The author’s research is supported by Public Health Service Grant GM056141 + dependent attenuation mechanism that acts as a Mg2 -dependent from the National Institutes of Health.

14882 | www.pnas.org/cgi/doi/10.1073/pnas.1618222114 Hughes Downloaded by guest on September 28, 2021 1 Crick F (1970) Central dogma of molecular biology. Nature 227(5258):561–563. 2 Kriner MA, Sevostyanova A, Groisman EA (2016) Learning from the leaders: Gene regulation by the transcription termination factor Rho. Trends Biochem Sci 41(8): 690–699. 3 Naville M, Gautheret D (2010) Transcription attenuation in bacteria: Theme and variations. Brief Funct Genomics 9(2):178–189. 4 Peters JM, Vangeloff AD, Landick R (2011) Bacterial transcription terminators: the RNA 3′-end chronicles. J Mol Biol 412(5):793–813. 5 Alifano P, Rivellini F, Limauro D, Bruni CB, Carlomagno MS (1991) A consensus motif common to all Rho-dependent prokaryotic transcription terminators. Cell 64(3):553–563. 6 Nudler E, Gottesman ME (2002) Transcription termination and anti-termination in E. coli. Genes Cells 7(8):755–768. 7 Landick R, Turnbough CL, Jr, Yanofsky C (1996) Transcription attenuation. Escherichia coli and Salmonella: Cellular and Molecular Biology, ed Neidhardt FC (ASM Press, Washington, DC), Vol 1, pp 1263–1286. 8 Breaker RR (2011) Prospects for riboswitch discovery and analysis. Mol Cell 43(6):867–879. 9 Mellin JR, Cossart P (2015) Unexpected versatility in bacterial riboswitches. Trends Genet 31(3):150–156. 10 Brenner M, Ames BN (1971) The histidine operon and its regulation. Metabolic Pathways, ed Vogel HS (Academic, New York), Vol 5, pp 349–387. 11 Washburn RS, Gottesman ME (2015) Regulation of transcription elongation and termination. Biomolecules 5(2):1063–1078. 12 Johnston HM, Barnes WM, Chumley FG, Bossi L, Roth JR (1980) Model for regulation of the histidine operon of Salmonella. Proc Natl Acad Sci USA 77(1):508–512. 13 Barnes WM (1978) DNA sequence from the histidine operon control region: Seven histidine codons in a row. Proc Natl Acad Sci USA 75(9):4281–4285. 14 Chevance FF, Le Guyon S, Hughes KT (2014) The effects of codon context on in vivo translation speed. PLoS Genet 10(6):e1004392. 15 Pavlov MY, et al. (2009) Slow peptide bond formation by proline and other N-alkylamino acids in translation. Proc Natl Acad Sci USA 106(1):50–54. 16 Doerfel LK, et al. (2013) EF-P is essential for rapid synthesis of proteins containing consecutive proline residues. Science 339(6115):85–88. + 17 Gall AR, et al. (2016) Mg2 regulates transcription of mgtA in Salmonella Typhimurium via translation of proline codons during synthesis of the MgtL peptide. Proc Natl Acad Sci USA 113:15096–15101. 18 Groisman EA (1998) The ins and outs of virulence gene expression: Mg2+ as a regulatory signal. BioEssays 20(1):96–101. 19 Monsieurs P, et al. (2005) Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium. J Mol Evol 60(4):462–474. 20 Papp-Wallace KM, Maguire ME (2008) Regulation of CorA Mg2+ channel function affects the virulence of Salmonella enterica serovar typhimurium. J Bacteriol 190(19):6509–6516. 21 Soncini FC, Garc´ıaV´escovi E, Solomon F, Groisman EA (1996) Molecular basis of the magnesium deprivation response in Salmonella typhimurium: Identification of PhoP-regulated genes. J Bacteriol 178(17):5092–5099.

Hughes PNAS | December 27, 2016 | vol. 113 | no. 52 | 14883 Downloaded by guest on September 28, 2021