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Journal of Bacteriology, September 2004, p . 6311-6315, Vol . 186, No . 18

mRNA Secondary Structure Modulates Translation of Tat-Dependent Formate Dehydrogenase N

Claire Punginelli,1 Bérengère Ize,1,2 Nicola R . Stanley,1,2,{dagger} Valley Stewart,3 Gary Sawers,1 Ben C . Berks,4 and Tracy Palmer1,2*

Department of Molecular Microbiology, John Innes Centre,1 School of Biological Sciences, University of East Anglia, Norwich,2 Department of Biochemistry, University of Oxford, Oxford, United Kingdom,4 Section of Microbiology, University of California, Davis, California3

Received 14 May 2004/ Accepted 14 June 2004


 

  ABSTRACT

 
Formate dehydrogenase N [FDH-N] of Escherichia coli is a membrane-bound enzyme comprising FdnG, FdnH, and FdnI subunits organized inan [{alpha}ß{gamma}]3 configuration . The FdnG subunit carries aTat-dependent signal peptide, which localizes the protein complexto the periplasmic side of the membrane . We noted that substitutionof the first arginine [R5] in the twin arginine signal sequenceof FdnG for a variety of other amino acids resulted in a dramatic[up to 60-fold] increase in the levels of protein synthesized.Bioinformatic analysis suggested that the mRNA specifying thefirst 17 codons of fdnG forms a stable stem-loop structure.A detailed mutational analysis has demonstrated the importanceof this mRNA stem-loop in modulating FDH-N translation.


 

  TEXT

 
The respiratory formate dehydrogenase N [FDH-N] enzyme of Escherichia coli is a seleno-molybdoenzyme that is synthesized when the bacterium grows anaerobically with nitrate as exogenous electron acceptor . FDH-N can comprise up to 10% of the total membraneprotein [9] . Together with nitrate reductase-A, it forms a respiratory chain transferring electrons from formate to nitrate and results in the generation of a protonmotive force [10] . FDH-N has anumber of cofactors, including bis-molybdopterin guanine dinucleotidecofactor, selenocysteine, and a single [4Fe-4S] cluster . Consequently,synthesis of this enzyme requires careful control . Transcriptionof both the fdnGHI operon, which encodes FDH-N, and narGHJI,which encodes nitrate reductase-A, is coordinately controlled.Expression of both operons is maximal anaerobically in the presenceof nitrate and is controlled by the transcription factors Fnrand NarL [8].

The high-resolution X-ray structure of FDH-N has revealed thatit adopts an [{alpha}ß{gamma}]3 "trimer-of-trimers " architecture,with the active site of the enzyme located in the periplasm[13] . FDH-N is translocated across the membrane by the twinarginine translocation [Tat] pathway [21] . The Tat translocaseis dedicated to the transport of prefolded proteins, which bearan N-terminal signal peptide with the conserved S/T-R-R-x-F-L-Ktwin arginine motif [2, 3] . Translocation of FDH-N is mediatedby virtue of a Tat signal peptide on the FdnG subunit . Stanleyet al . have shown previously that the FdnG signal peptide isable to mediate export of the reporter proteins ß-lactamaseand chloramphenicol acetyltransferase to the periplasm in aTat-dependent fashion [24] . During the course of these fusionstudies we noted that point mutations in the first or secondarginine codons of the twin arginine motif resulted in a dramaticoverproduction of the fusion protein . The mRNA specifying thefirst 17 codons of the fdnG gene is predicted to fold into astable stem-loop structure, and we demonstrate here that this stem-loop mediates translational control of FDH-N synthesis.

As shown in Table 1, substitution of Arg5, either conservativelyfor Lys or nonconservatively for Ser, resulted in a marked increase[up to 60-fold] in fusion protein synthesis, regardless of thenature of the reporter protein . In contrast, substitution ofLys10 for Glu had no significant effect on levels of fusionprotein [Table 1] . No significant differences in fusion proteinsynthesis were noted in a tat mutant background, suggestingthat these observations were not directly related to operationof the Tat pathway . When the mfold program [25] was used, themRNA covering the start codon and signal peptide-coding regionof fdnG could be folded into a stem-loop structure [Fig . 1B].The {Delta}G value [at 37°C and physiological pH] associated withthis structure was calculated to be –12.6 kCal/mol, suggestingthat the folded mRNA would be relatively stable . Such a foldedmRNA structure would be consistent with the results seen inTable 1, since mutations at codon 5, which fall within one armof the predicted stem, would be expected to disrupt the fold,whereas the mutation at codon 10, which is located within theputative loop region, would not be expected to disrupt the structure.


 

TABLE 1 . Total cellular activity of reporter proteins fused to the wild-type or amino acid-substituted FdnG signal sequencea

 

 

FIG . 1 . The mRNA specifying the fdnG signal peptide coding region can be folded into a stem-loop structure . The position of the initiation codon is indicated in bold type; the Shine-Dalgarno ribosome-binding sequence is underlined . Numbering is shown relative to the first base of the start codon, which is designated +1 . [A] Portion of the DNA from plasmid pVJS2248 [24] showing the first part of the fdnG signal peptide-coding region . To aid clarity, the amino acids are numbered with subscripts . The amino acids of the twin arginine motif are doubly underlined . The clone carries a 487-bp insert that covers the entire fdnG promoter region and fdnG sequence as far as an engineered BamHI site in the DNA after codon 44 [24] . [B] The mRNA covering the region shown in panel A was folded using the program mfold 3 [25], which was accessed through the world-wide web [http://www. bioinfo.rpi.edu /applications/mfold] . The codon substitutions used during this study are V3P [GTC->CCC], R5K [CGC->AAG], R5S [CGC->AGC], R5Rhigh [CGC->CGT], R5Rlow [CGC->AGG], R6K [AGA->AAG], R6Rhigh [AGA->CGT], F9L [TTT->CTG], K10E [AAA->GAA], I11V [ATC->GTC], I11I [ATC->ATA], I11T [ATC->ACT], C12A [TGC->GCC], A13S [GCG->AGC], G14K [GGC->AAA], and R6K/I11T [AGA->AAG and ATC->ACT].

 
To test whether a hairpin in the fdnG mRNA was controlling expression at a translational level, we constructed a number of additional mutations in codons 3 to 14 of fdnG . As shown in Fig . 2A, substitutionsthat were predicted to severely disrupt the mRNA fold [R5S [CGC->AGC], R5K [CGC->AAG], R5Rhigh [CGC->CGT], and R5Rlow [CGC->AGG], where thenatural Arg codon was replaced with Arg codons of higher andlower usage] [18] led to significant upregulation of ß-galactosidaseactivity . Mutations in codon 6 that were predicted to have moremodest effects on the mRNA secondary structure gave less-dramaticincreases in ß-galactosidase activity . As predictedby the model, substitutions of codons that fell within the putativeloop region [Fig . 2B] did not have significant effects on theactivity of ß-galactosidase, with the exception ofthe I11V mutation, which resulted in a marked decrease in theactivity of ß-galactosidase . Interestingly, this substitution is predicted to result in a significant increase in the stability of the stem-loop [from –12.6 to –16.4 kCal/mol]due to the formation of an extra base pair at the top of thestem . The results of substitutions in the putative second armof the stem [Fig. 2C] were also consistent with the premisethat a stem-loop structure was controlling translation of fdnG-lacZ. A plot of {Delta}G value for the structures associated with the wild-typesequence and each of the mutations against the observed ß-galactosidaseactivity [Fig . 2E] shows a linear relationship . Indeed, a straight line can be drawn through the points with a correlation factor [R2] of 0.82, providing strong support for the model . As a finaltest, we looked at the effect of introducing a compensatory mutation into the second arm of the stem, to restore base pairing, with a mutation in the first arm of the stem that disrupts the stem-loop structure . When introduced singly, both the AGA->AAG mutations at codon 6 and the ATC->ACT mutations at codon 11 ledto increased ß-galactosidase activity [Fig. 2D] . However,when these two sets of mutations were combined, ß-galactosidaseactivity was restored to the wild-type level, indicating thatthe two mutations had a compensatory effect.


 

FIG . 2 . The effects of mutations in the stem and loop regions of the fdnG mRNA on expression of fdnG-lacZ . Constructs were transformed into strain MC4100-P [4], which carries the pcnB allele, providing more careful control of plasmid copy number [14, 15] . [A] Constructs carrying mutations in the codons forming the first arm of the stem; [B] constructs carrying mutations in codons that fall in the loop region; [C] constructs carrying mutations in codons forming the second arm of the stem; [D] constructs carrying either individual single codon substitutions or a combined double-codon substitution that is predicted to have a compensatory effect . All strains were cultured anaerobically in Cohen and Rickenberg medium [7] supplemented with 0.2% glucose and 0.4% nitrate . ß-Galactosidase enzyme activity is shown in Miller units [17], and the bars represent standard errors of the means [n = 3 to 4] . [E] Correlation between stem-loop stability [measured as {Delta}G] and ß-galactosidase enzyme activity . Free energies for each codon mutation were calculated with the program mfold3 [25] using refined energy parameters [16] and with the mRNA sequence covering codons 1 to 17 inclusive . WT, wild type.

 
In addition to the substantial transcriptional regulation already reported [1], our findings strongly suggest that a further, possibly up to 10-fold, level of control of fdnGHI expression was exerted at the translational level . To confirm the physiological relevance of these findings, we used published methods [11] to introduce two sets of mutations [R5Rhigh [CGC->CGT] and R5Rlow[CGC->AGG]] in the fdnG gene in the chromosome . In each case wemade a substitution that retained an Arg codon at position 5so that the cellular location of FDH-N would not be compromised.To circumvent possible translational errors associated withuse of rare arginine codons, such as AGG, in E . coli [see, e.g.,reference 5], we additionally introduced into each strain plasmid pUt-AGA/AGG, which carries the genes encoding tRNAArgUCU andtRNAArgUCC [22] . In the presence of this plasmid, but not inits absence, we saw an approximate fourfold increase in FDH-Nactivity from the strain with the R5Rlow chromosomal replacementof fdnG [Fig. 3A] and a corresponding overproduction of theFdnG polypeptide [Fig . 3C] . This result strongly suggests that the translational control of fdnG expression is of physiological significance, since we have clearly demonstrated that the cell has the capacity to synthesize, assemble, and export fourfold-higher levels of FDH-N . Interestingly, as shown in Fig . 3B the fourfoldoverproduction of FDH-N activity was associated with a markedincrease in NAR activity . This suggests that there might be a further level of coordinate control of FDH-N and NAR synthesis not previously observed.


 

FIG . 3 . The stem-loop structure regulates FDH-N activity in vivo . FDH-N [A] and NAR [B] enzyme assays were performed with crude cell extracts derived from strains MC4100, NRS9 [as MC4100; FdnG R5R [CGC->CGT]], and CMP1 [as MC4100; FdnG R5R [CGC->AGG]] or the same strains carrying plasmid pUt-AGA/AGG [22] . Cells were grown anaerobically in Luria-Bertani medium supplemented with 0.2% glucose and 0.4% potassium nitrate, crude extracts were prepared, and enzyme activities were assayed exactly as described previously [21] . Assays were performed in triplicate with results differing by not more than 15% of the mean . [A] FDH-N enzyme activity [measured as the rate of phenazine methosulfate-linked formate dehydrogenase activity] [9] . An activity level of 100% is that measured from the crude cell extract of the wild-type [WT] strain and corresponds to 33 nmol of formate oxidized/min/mg of protein . [C] NAR enzyme activity, measured according to the method of Jones and Garland [12] . An activity level of 100% is that measured in the crude cell extract of the wild-type strain and corresponds to 598 nmol of nitrate reduced/min/mg of protein . [C] Detection of FdnG polypeptide in whole cells after Western blot analysis with anti-FdnG antibodies [24] . Equivalent quantities of cells were loaded in each lane . The fold overexpression of FdnG polypeptide relative to that detected in the wild-type sample is shown above each lane.

 
In conclusion, we have demonstrated that there is control ofFDH-N synthesis at the translational level . Interestingly, thestem-loop structure seen here for the E . coli mRNA is also predictedto be conserved in the fdnG coding regions of several other bacteria, including Salmonella enterica serovar Typhimurium, Haemophilus influenzae, and Pseudomonas aeruginosa . Notably, however, the stem-loop structure is apparently not conserved over the similar coding region of E . coli fdoG . While the mechanism underlying modulation of FDH-N translation is presently unclear, it may potentially serve as a quality control feature ensuring that only the fully assembled complex is transported to the periplasm.

 


 

  ACKNOWLEDGMENTS

 
This work was supported by a John Innes Foundation Studentship[to C.P.], by a Norwich Research Park Studentship [to N.R.S.],and by the BBSRC via grant funding and a grant-in-aid to theJohn Innes Centre . Work in the Stewart laboratory was supportedby Public Health Service grant GM36877 from the National Instituteof General Medical Sciences . T.P . is a Royal Society ResearchFellow.

We thank Jiarong Shi for her essential contributions to the initial stages of this study and Frank Sargent for helpful discussion.


 

  FOOTNOTES

 
* Corresponding author . Mailing address: Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom . Fax: [44] [1603] 450778 . E-mail: tracy.palmer@bbsrc.ac.uk.

 

{dagger} Present address: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095-1489.


 

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