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Stem-loop structures predicted downstream of SBPs. (A) Stem-loops predicted in gene clusters encoding ABC importers from R. cellulolyticum, which were classified into four types based on their structural features. Putative 39 U-rich sequences are shown in gray boxes. (B and C) Comparison of the RNA folding free energy (DG) (B) and 39 U content (C) of four types of stem-loop structures from all eight Clostridia.
Source publication
Operons encoding protein complexes or metabolic pathways usually require fine-tuned gene expression ratios to create and maintain the appropriate stoichiometry for biological functions. In this study, a strategy for controlling differential expression of genes in an operon was proposed by utilizing ABC importers from Ruminiclostridium cellulolyticu...
Contexts in source publication
Context 1
... Thus, sequences at the 39 paired or unpaired U-rich tails for Type I and Type II stem-loops and downstream of unpaired regions in Types III and IV were considered to be putative 39 U-rich tracts of Rho-independent terminators (Table S2). For instance, R. cellulolyticum had two Type I, five Type II, five Type III, and four Type IV stem-loops (Fig. 4A). The differences in the structure of stem-loops led to differences in their folding free energy. Type IV stem-loop structures had the lowest average folding free energy at 228.4 kcal/mol, while Type I had the highest, at 218.3 kcal/mol (Fig. 4B). In contrast, the results of comparisons of U content of 39 Urich tracts indicated that ...
Context 2
... instance, R. cellulolyticum had two Type I, five Type II, five Type III, and four Type IV stem-loops (Fig. 4A). The differences in the structure of stem-loops led to differences in their folding free energy. Type IV stem-loop structures had the lowest average folding free energy at 228.4 kcal/mol, while Type I had the highest, at 218.3 kcal/mol (Fig. 4B). In contrast, the results of comparisons of U content of 39 Urich tracts indicated that the U content of the 39 U-rich tracts of Type I stem-loops ranged from 5 to 8 as the highest, while Type IV stem-loops tended to be the lowest, as most of these hardly had obvious U-rich regions (Fig. 4C). Given the structural features of these ...
Context 3
... while Type I had the highest, at 218.3 kcal/mol (Fig. 4B). In contrast, the results of comparisons of U content of 39 Urich tracts indicated that the U content of the 39 U-rich tracts of Type I stem-loops ranged from 5 to 8 as the highest, while Type IV stem-loops tended to be the lowest, as most of these hardly had obvious U-rich regions (Fig. 4C). Given the structural features of these stem-loops and the transcriptional profiles of ABC importers, we speculated that these stem-loops could cause transcription to terminate as internal transcriptional ...
Citations
... The role of RNase J in the cip-cel mRNA was further confirmed by the rnj mutant, in which the transcript level of the cip-cel operon was much higher than wild-type, suggesting that RNase J contributes to the stability of the cip-cel mRNA (Additional file 1: Fig. S4). Furthermore, the structure of the stem-loops at the 3′ end of processed transcripts, such as IR1-SL1, IR2-SL, IR5-SL1, IR7-SL1 and IR10-SL, is more complex than those at the 5′ end, including IR1-SL2, IR4-SL, IR5-SL2, and IR7-SL2, that is consistent with our previous observations of stronger protection of the 3′ end in R. cellulolyticum [9,50]. Together, the structural difference of stem-loops at both ends of processed transcripts suggested that the activity of 3′-to-5′ exoribonucleases was much higher than that of 5′-to-3′ exoribonucleases in R. cellulolyticum, resulting in stronger protection at the 3′ end of processed transcripts. ...
Background
Anaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.
Results
In this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3′ end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon.
Conclusions
Our findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.
Mesophilic, anaerobic, and cellulolytic Ruminiclostridium -type bacterial species can secrete an extracellular, multi-enzyme machinery cellulosome, which efficiently degrades cellulose. In this study, we first reported the complete genome of Ruminiclostridium papyrosolvens DSM2782, a single circular 5,027,861-bp chromosome with 37.1% G + C content, and compared it with other Ruminiclostridium -type species. Pan-genome analysis showed that Ruminiclostridium -type species share a large number of core genes to conserve basic functions, although they have a high level of intraspecific genetic diversity. Especially, KEGG mapping revealed that Ruminiclostridium -type species mainly use ABC transporters regulated by two-component systems (TCSs) to absorb extracellular sugars but not phosphotransferase systems (PTSs) that are employed by solventogenic clostridia, such as Clostridium acetobutylicum . Furthermore, we performed comparative analyses of the species-specific repertoire of CAZymes for each of the Ruminiclostridium -type species. The high similarity of their cohesins suggests a common ancestor and potential cross-species recognition. Additionally, both differences between the C-terminal cohesins and other cohesins of scaffoldins and between the dockerins linking with cellulases and other catalytic domains indicate a preference for the location of cellulosomal catalytic subunits at scaffoldins. The information gained in this study may be utilized directly or developed further by genetic engineering and optimizing enzyme systems or cell factories for enhanced biotechnological biomass deconstruction and biofuel production.
Genes organized within operons in prokaryotes benefit from coordinated expression. However, within many operons, genes are expressed at different levels, and the mechanisms for this remain obscure. By integrating PacBio-seq, dRNA-seq, Term-seq and Illumina-seq data of a representative archaeon Methanococcus maripaludis, internal transcription termination sites (ioTTSs) were identified within 38% of operons. Higher transcript and protein abundances were found for genes upstream than downstream of ioTTSs. For representative operons, these differences were confirmed by northern blotting, qRT-PCR and western blotting, demonstrating that these ioTTS terminations were functional. Of special interest, mutation of ioTTSs in ribosomal protein (RP)-RNA polymerase (RNAP) operons not only elevated expression of the downstream RNAP genes but also decreased production of the assembled RNAP complex, slowed whole cell transcription and translation, and inhibited growth. Overexpression of the RNAP subunits with a shuttle vector generated the similar physiological effects. Therefore, ioTTS termination is a general and physiologically significant regulatory mechanism of the operon gene expression. Because the RP-RNAP operons are found to be widely distributed in archaeal species, this regulatory mechanism could be commonly employed in archaea.
