Conditional Switch between Frameshifting Regimes upon Translation of dnaX mRNA.

Ribosome frameshifting during translation of bacterial dnaX can proceed via different routes, generating a variety of distinct polypeptides. Using kinetic experiments, we show that -1 frameshifting predominantly occurs during translocation of two tRNAs bound to the slippery sequence codons. This pathway depends on a stem-loop mRNA structure downstream of the slippery sequence and operates when aminoacyl-tRNAs are abundant. However, when aminoacyl-tRNAs are in short supply, the ribosome switches to an alternative frameshifting pathway that is independent of a stem-loop. Ribosome stalling at a vacant 0-frame A-site codon results in slippage of the P-site peptidyl-tRNA, allowing for -1-frame decoding. When the -1-frame aminoacyl-tRNA is lacking, the ribosomes switch into -2 frame. Quantitative mass spectrometry shows that the -2-frame product is synthesized in vivo. We suggest that switching between frameshifting routes may enrich gene expression at conditions of aminoacyl-tRNA limitation.

Protein Chemical Modification Inside Living Cells Using Split Inteins.

Methods to visualize, track, measure, and perturb or activate proteins in living cells are central to biomedical efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We describe in this protocol the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split-inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins in order to control their cellular localization and potentially alter their biological activity.

Systematic identification of synthetic lethal mutations with reduced-genome Escherichia coli: synthetic genetic interactions among yoaA, xthA and holC related to survival from MMS exposure.

Reduced-genome Escherichia coli strains lacking up to 38.9% of the parental chromosome have been constructed by combining large-scale chromosome deletion mutations. Functionally redundant genes involved in essential processes can be systematically identified using these reduced-genome strains. One large-scale chromosome deletion mutation could be introduced into the wild-type strain but not into the largest reduced-genome strain, suggesting a synthetic lethal interaction between genes removed by the deletion and those already absent in the reduced-genome strain. Thus, introduction of the deletion mutation into a series of reduced-genome mutants could allow the identification of other chromosome deletion mutations responsible for the synthetic lethal phenotype. We identified a synthetic lethality caused by disruption of nfo and xthA, two genes encoding apurinic/apyrimidinic (AP) endonucleases involved in the DNA base excision repair pathway, and two other large-scale chromosome deletions. We constructed temperature-sensitive mutants harboring quadruple-deletion mutations in the affected genes/chromosome regions. Using these mutants, we identified two multi-copy suppressors: holC, encoding the chi subunit of DNA polymerase III, and yoaA, encoding a putative DNA helicase. Addition of the yoaA disruption increased the methyl methanesulfonate (MMS) sensitivity of xthA single-deletion or xthA nfo double-deletion mutants. This increased MMS sensitivity was not suppressed by the presence of multi-copy holC. These results indicate that yoaA is involved in MMS sensitivity and suggest that YoaA functions together with HolC.

The multifaceted benefits of protein co-expression in Escherichia coli.

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3'-5' exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-ß-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Biogenesis of multisubunit RNA polymerases.

Gene transcription in the nucleus of eukaryotic cells is carried out by three related multisubunit RNA polymerases, Pol I, Pol II and Pol III. Although the structure and function of the polymerases have been studied extensively, little is known about their biogenesis and their transport from the cytoplasm (where the subunits are synthesized) to the nucleus. Recent studies have revealed polymerase assembly intermediates and putative assembly factors, as well as factors required for Pol II nuclear import. In this review, we integrate the available data into a model of Pol II biogenesis that provides a framework for future analysis of the biogenesis of all RNA polymerases.

Production of recombinant human DNA polymerase delta in a Bombyx mori bioreactor.

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability.

Production of Nα-acetylated thymosin α1 in Escherichia coli.

BACKGROUND: Thymosin α1 (Tα1), a 28-amino acid Nα-acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining Nα-acetylation. In this study, we describe a novel production process for Nα-acetylated Tα1 in Escherichia coli. RESULTS: To obtain recombinant Nα-acetylated Tα1 efficiently, a fusion protein, Tα1-Intein, was constructed, in which Tα1 was fused to the N-terminus of the smallest mini-intein, Spl DnaX (136 amino acids long, from Spirulina platensis), and a His tag was added at the C-terminus. Because Tα1 was placed at the N-terminus of the Tα1-Intein fusion protein, Tα1 could be fully acetylated when the Tα1-Intein fusion protein was co-expressed with RimJ (a known prokaryotic Nα-acetyltransferase) in Escherichia coli. After purification by Ni-Sepharose affinity chromatography, the Tα1-Intein fusion protein was induced by the thiols ß-mercaptoethanol or d,l-dithiothreitol, or by increasing the temperature, to release Tα1 through intein-mediated N-terminal cleavage. Under the optimal conditions, more than 90% of the Tα1-Intein fusion protein was thiolyzed, and 24.5 mg Tα1 was obtained from 1 L of culture media. The purity was 98% after a series of chromatographic purification steps. The molecular weight of recombinant Tα1 was determined to be 3107.44 Da by mass spectrometry, which was nearly identical to that of the synthetic version (3107.42 Da). The whole sequence of recombinant Tα1 was identified by tandem mass spectrometry and its N-terminal serine residue was shown to be acetylated. CONCLUSIONS: The present data demonstrate that Nα-acetylated Tα1 can be efficiently produced in recombinant E. coli. This bioprocess could be used as an alternative to chemosynthesis for the production of Tα1. The described methodologies may also be helpful for the biosynthesis of similar peptides.

Characterization and comparison of chicken U6 promoters for the expression of short hairpin RNAs.

RNA interference (RNAi) is a powerful method of sequence-specific gene knockdown that can be mediated by DNA-based expression of short hairpin RNA (shRNA) molecules. A number of vectors for expression of shRNA have been developed with promoters for a small group of RNA polymerase III (pol III) transcripts of either mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and future development of specific therapeutics in the chicken species, we have developed shRNA expression vectors featuring chicken U6 small nuclear RNA (snRNA) promoters. These sequences were identified based on the presence of promoter element sequence motifs upstream of matching snRNA sequences that are characteristic of these types of pol III promoters. To develop suitable shRNA expression vectors specifically for chicken functional genomic RNAi applications, we compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and silencing the reporter gene encoding the enhanced green fluorescent protein (EGFP). Plasmids containing one of four identified chicken U6 promoters gave a similar degree of knockdown in DF-1 cells (chicken); although, there was some variability in Vero cells (monkey). Because the chicken promoters were not stronger than the benchmark mouse U6 promoter, we suggest that the promoter sequence and structure is more important in determining efficiency in vitro rather than its species origin.

Expression of split dnaE genes and trans-splicing of DnaE intein in the developmental cyanobacterium Anabaena sp. PCC 7120.

Protein intein is widespread in a variety of organisms. Several intein elements are also present in cyanobacteria, and some of them have been studied biochemically in vitro. However, no evidence is available for intein removal in vivo in cyanobacteria. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the DNA replication factor DnaE is encoded by two split open reading frames (ORFs) far apart from each other on the chromosome, and each of them could contain a split intein element. This organism can undergo a developmental process leading to the formation of nitrogen-fixing cells, or heterocysts. Heterocysts are terminally differentiated cells with arrest of cell cycle. Since DnaE is an important cell cycle element involved in DNA replication, we would like to provide in vivo evidence for DnaE intein removal in cyanobacteria and determine whether mature DnaE protein is still present in heterocysts. In this study, we showed that the products of these two ORFs were joined together to form a complete DnaE protein through the process of protein trans-splicing. More interestingly, protein trans-splicing could be detected in vivo for the first time in cyanobacteria, which allowed us to compare the formation of mature DnaE protein in heterocysts and vegetative cells, and show that mature DnaE protein could be formed in both cell types. Transcriptional fusion between the promoter regions of the two split ORFs and gfp reporter also demonstrate that both ORFs are transcribed in vegetative cells and heterocysts, without strong variation during the process of heterocyst differentiation. Although heterocysts are terminally differentiated and may not replicate its chromosome, the expression and maturation of DnaE in these cells may underlie the need for DNA replication machinery in processes such as DNA recombination and repair.

FGF2-induced upregulation of DNA polymerase-delta p12 subunit in endothelial cells.

p12 represents the smallest, so far poorly characterized subunit of the mammalian DNA polymerase delta (pol delta) heterotetramer. Previously, to gain a molecular understanding of endothelial cell activation by fibroblast growth factor-2 (FGF2), we identified an upregulated transcript in FGF2-overexpressing murine aortic endothelial cells (FGF2-T-MAE cells) showing 89% identity with human p12. Here, we cloned the open reading frame of the murine p12 cDNA and confirmed the capacity of overexpressed or exogenously added FGF2 to upregulate p12 mRNA and protein in endothelial and NIH3T3 cells with no effect on the other pol delta subunits. p12 expression was instead unaffected by serum and different mitogens. Also, anti-p12 antibodies decorated FGF2-T-MAE cell nuclei and their chromosome outline during metaphase. Small interfering RNA-mediated knockdown of p12 caused a significant decrease in FGF2-driven proliferation rate of FGF2-T-MAE cells, in keeping with a modulatory role of p12 in pol delta activity. Immunoistochemistry of FGF2-embedded Matrigel plugs and FGF2-overexpressing tumor xenografts demonstrated a nuclear p12 staining of angiogenic CD31(+) endothelium. p12 immunoreactivity was also observed in the CD45(+)/CD11b(+) inflammatory infiltrate. Thus, FGF2 upregulates p12 expression in endothelial cells in vitro and in vivo. p12 expression in infiltrating inflammatory cells may suggest additional, cell proliferation-unrelated functions for this pol delta subunit.

Effect of exogenous MSH6 and POLD1 expression on the mutation rate of the HPRT locus in a human colon cancer cell line with mutator phenotype, DLD-1.

The DLD-1 human colon cancer cell line displays an elevated spontaneous mutation rate. Since DLD-1 carries frameshift mutations in both alleles of the MSH6 gene and missense mutations in the POLD1 gene, either or both of these mutations were suggested to be involved in this mutator phenotype. Therefore, we examined the effect of exogenous wild-type MSH6 and POLD1 expression on the spontaneous mutation rate at the HPRT locus in DLD-1 cells. POLD1 genotypes were first determined, since four POLD1 missense mutations were previously reported in DLD-1 cells. Sequencing analyses on the genomic DNA and cDNA of the POLD1 gene revealed that DLD-1 cells are a mixture of two distinct sublines with regard to POLD1 genotypes. Moreover, the wild-type POLD1 allele was not present in either of the two DLD-1 sublines. We next established MSH6- and POLD1-transfected DLD-1 clones from both sublines, respectively. The two DLD-1 sublines exhibited HPRT mutation rates of 4.8 x 10(-6) and 5.4 x 10(-6) mutations/cell/generation. The mutation rates were more than 4-fold decreased in both of the MSH6-transfected DLD-1 clones examined, while they were not significantly decreased in three of four POLD1-transfected DLD-1 clones. Thus, it was indicated that mutations in the MSH6 gene, and not in the POLD1 gene, are primarily responsible for the elevated mutation rates in DLD-1 cells.

DNA polymerase III holoenzyme from Thermus thermophilus identification, expression, purification of components, and use to reconstitute a processive replicase.

DNA replication in bacteria is performed by a specialized multicomponent replicase, the DNA polymerase III holoenzyme, that consist of three essential components: a polymerase, the beta sliding clamp processivity factor, and the DnaX complex clamp-loader. We report here the assembly of the minimal functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme consists of alpha (pol III catalytic subunit), beta (sliding clamp processivity factor), and the essential DnaX (tau/gamma), delta and delta' components of the DnaX complex. We show with purified recombinant proteins that these five components are required for rapid and processive DNA synthesis on long single-stranded DNA templates. Subunit interactions known to occur in DNA polymerase III holoenzyme from mesophilic bacteria including delta-delta' interaction, deltadelta'-tau/gamma complex formation, and alpha-tau interaction, also occur within the Tth enzyme. As in mesophilic holoenzymes, in the presence of a primed DNA template, these subunits assemble into a stable initiation complex in an ATP-dependent manner. However, in contrast to replicative polymerases from mesophilic bacteria, Tth holoenzyme is efficient only at temperatures above 50 degrees C, both with regard to initiation complex formation and processive DNA synthesis. The minimal Tth DNA polymerase III holoenzyme displays an elongation rate of 350 bp/s at 72 degrees C and a processivity of greater than 8.6 kilobases, the length of the template that is fully replicated after a single association event.

Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core.

Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.

DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%.

BACKGROUND AND AIMS: We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. METHODS: The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase alpha, delta, and epsilon were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. RESULTS: The 90% hepatectomy resulted in the strong inductions of DNA polymerase alpha, delta, and epsilon, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. CONCLUSION: The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma.

Ara-C affects formation of cancer cell DNA synthesome replication intermediates.

PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.

Molecular cloning and characterization of the murine staf cDNA encoding a transcription activating factor for the selenocysteine tRNA gene in mouse mammary gland.

We have isolated and characterized a cDNA encoding a transcription activating factor for the mouse selenocysteine tRNA (tRNAsec) gene from mouse mammary gland. The full-length cDNA, designated m-Staf, has a 1878-base pair open reading frame encoding 626 amino acids. The predicted amino acid sequence of m-Staf is highly homologous to that of Staf, another selenocysteine tRNA gene transcription activating factor of Xenopus laevis. Like Staf, m-Staf contains seven tandemly repeated zinc fingers and four repeated motifs. Gel shift assays indicated that the recombinant m-Staf specifically bound to the activator element region in the mouse tRNAsec gene. Transient co-transfection experiments in Drosophila Schneider cells, which lack endogenous Staf-like binding activity, showed that m-Staf increased the mouse tRNAsec gene transcription about 15-fold, whereas it stimulated Pol II-dependent thymidine kinase promoter only 2-fold. Northern blot analysis detected the presence of a 3.4-kilobase pair m-Staf transcript, which was widely but differentially expressed in various murine tissues. The binding activity of m-Staf in mouse mammary gland was undetectable during virgin and postlactating periods but increased markedly in parallel with the increase of tRNAsec transcript during the periods of pregnancy and lactation, when the gland undergoes growth and development. These results indicate that m-Staf is a transcriptional activator of the mouse tRNAsec gene and that its binding activity in the mammary gland undergoes developmental alterations.

Thermus thermophilis dnaX homolog encoding gamma- and tau-like proteins of the chromosomal replicase.

This report identifies the dnaX homolog from Thermus thermophilis. Replicases from bacteria to humans contain subunits that are homologous to one another. These homologs are subunits of a clamp loading apparatus that loads sliding clamps onto DNA, which in turn act as mobile tethers for the replication machinery. In Escherichia coli, two of these subunits (gamma and tau) are encoded by one gene (dnaX) in nearly equal amounts by way of an efficient translational frameshift. The gamma and tau subunits form the central touchpoint that holds together two DNA polymerases with one clamp loading apparatus to form the E. coli chromosomal replicase, DNA polymerase III holoenzyme. The E. coli holoenzyme is an efficient replication machine that simultaneously replicates both strands of duplex DNA. The T. thermophilis dnaX homolog also contains a frameshift signature and produces both tau- and gamma-like proteins. Recombinant T. thermophilis tau- and gamma-like proteins, expressed in E. coli, have an oligomeric state similar to that of their E. coli counterparts and display ATPase activity that is stimulated by DNA. These results imply that T. thermophilis utilizes a DNA polymerase III holoenzyme replication machinery similar to that of E. coli.

Structural probing and mutagenic analysis of the stem-loop required for Escherichia coli dnaX ribosomal frameshifting: programmed efficiency of 50%.

Three elements are crucial for the programmed frameshifting in translation of dnaX mRNA: a Shine-Dalgarno (SD)-like sequence, a double-shift site, and a 3' structure. The conformation of the mRNA containing these three elements was investigated using chemical and enzymatic probes. The probing data show that the structure is a specific stem-loop. The bottom half of the stem is more stable than the top half of the stem. The function of the stem-loop was further investigated by mutagenic analysis. Reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. Stabilizing the top half of the stem gives increased frameshifting beyond the WT efficiency. The identity of the primary RNA sequence in the stem-loop is unimportant, provided that the overall structure is maintained. The calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. The SD-interaction and the stem-loop element act independently to increase frameshifting in dnaX.