Method for Quantification of Rho-Dependent Termination Efficiency in vivo

Abstract

The present invention provides a method for measuring transcription termination efficiency that can be usefully used to calculate Rho-dependent termination efficiency in vivo, and a method for identifying an in vivo Rho-dependent termination site using the same. More specifically, in an operon sequence containing n transcription termination sites, a method for measuring the in vivo transcription termination efficiency at the i-th transcription termination site, transcription termination i (where n is a positive integer and i is a positive integer less than or equal to n), comprising: (A) a step of preparing a tRNA reporter gene having orthogonality with the tRNA of an experimental strain; (B) a step of constructing plasmids up and down by transcriptionally fusing the tRNA reporter gene with a gene up having a sequence (upstream of the operon's promoter to upstream of transcription termination i) or a gene down having a sequence (upstream of the operon's promoter to downstream of transcription termination i), respectively; (C) a step of producing recombinant strains up and down by transforming the above plasmid up and plasmid down into experimental strains, respectively; and (D) a step of quantifying the expression of tRNA reporter genes in vivo in the above recombinant strains up and down, respectively; the present invention relates to a method for measuring transcription termination efficiency in vivo and a method for identifying Rho-dependent termination sites in vivo using the same.

Description

Technology Field

[0001] The present invention aims to provide a method for measuring transcriptional termination efficiency that can be usefully used to calculate Rho-dependent termination efficiency in vivo, and a method for identifying the in vivo Rho-dependent termination site using the same. Background Technology

[0002] In bacteria, two forms of transcription termination procedures have been reported: Rho-independent termination (RIT) and Rho-dependent termination (RDT). The Rho-independent terminator, which induces Rho-independent termination, consists of two main modules: a guanine and cytosine-rich RNA stem loop structure and a 7-8 bp uridine-rich downstream sequence called the U tract. When the U tract is transcribed, it halts RNA polymerase (RNAP) for a sufficiently long period to allow for stem-loop formation, thereby disrupting the transcription complex and consequently terminating transcription at the U tract. Since the sequence characteristics of the Rho-independent terminator are well-defined and transcription termination occurs at the U-shaped tract, it is not difficult to determine the location of the Rho-independent terminator and detect Rho-independent termination in vivo.

[0003] Rho is a cyclic homohexameric protein with RNA-dependent ATPase activity. The Rho protein binds to a specific sequence of the mRNA transcript and moves along the RNA chain to the 3'-terminus, where it causes RNA polymerase (RNAP) to dissociate from the template DNA strand, thereby terminating transcription. In E. coli, it is known that approximately 20–30% of genes are terminated by Rho. Although many researchers have studied Rho-dependent termination through genetic and biochemical experiments for decades, there are still difficulties in predicting and / or detecting Rho-dependent termination in vivo for the following reasons: (1) The mechanism of Rho-dependent termination is complex, involving multiple components including RNAP, the initial RNA transcript, and the Rho protein, and the interactions between these components make it more difficult to understand the mechanism of Rho-dependent termination; (2) Rho is not specific to a particular mRNA sequence, and cytosine-rich Rho utilization ( rut (3) The Rho-dependent terminator consists of two main modules: a cytosine-rich and guanine-poor Rho binding region (C-rich region) lacking secondary structure and a termination region downstream of the C-rich region. The 3'-terminus of the mRNA is structurally unstable due to the lack of secondary structure and is rapidly degraded by 3'-5' exonucleases after Rho-dependent termination.

[0004] Quantitative real-time PCR (qRT-PCR) is a technique used to determine the amount of mRNA in a sample that can serve as an indicator of gene expression. It is utilized in various fields, including gene expression profiling, validation of gene expression arrays, disease diagnosis, new drug development, and forensic analysis. Chhakchhuak and Sen identified Rho-dependent termination sites using qRT-PCR. To this end, they designed multiple primer probes and suggested that if gene expression in Rho-deficient strains was upregulated when compared to wild-type strains, it implies the presence of a Rho-dependent termination site upstream of the corresponding probe location. Guenin S et al. and Minshall N et al. have reported that in qRT-PCR, the amounts of two different transcripts cannot be directly compared due to biases in reverse transcription and PCR amplification. Therefore, the above method can only confirm the difference in relative gene expression between wild-type strains and Rho-deficient strains, but cannot directly compare the difference in the amount of relative transcription upstream and downstream of the Rho termination site. If the amount of transcription upstream and downstream of a Rho-dependent termination site can be compared, transcription termination can be confirmed more directly and used to calculate transcription termination efficiency, which would be useful for regulating gene expression. Prior art literature

[0005] Chhakchhuak PIR, Sen R. 2022. In vivo regulation of bacterial Rho-dependent transcription termination by the nascent RNA. J Biol Chem 298:102001.Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L. 2009. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60:487-493.Minshall N, Git A. 2020. Enzyme- and gene-specific biases in reverse transcription of RNA raise concerns for evaluating gene expression. Sci Rep 10:8151.Nadiras 등, 2018, A multivariate prediction model for Rho dependent termination of transcription. Nucleic Acids Res 46:8245-8260.Di Salvo 등, 2019. RhoTermPredict: an algorithm for predicting Rho dependent transcription terminators based on Escherichia coli, Bacillus subtilis and Salmonella enterica databases. BMC Bioin-formatics 20:117. 해결하려는 과제

[0006] The present invention aims to solve the problems of the prior art and provide a method for directly and quantitatively measuring transcription efficiency by Rho-dependent termination in vivo.

[0007] In addition, another objective of the present invention is to provide a method for identifying a Rho-dependent termination site in vivo by applying the above method.

[0008] The technical problem that the present invention aims to solve will be clearly understood by those skilled in the art, even if not mentioned above.

[0009] In addition, when describing the invention, if it is determined that a detailed description of known technology related to the invention could unnecessarily obscure the essence of the invention, such detailed description will be omitted. means of solving the problem

[0010] The present invention, for achieving the aforementioned objective, is characterized by measuring transcription termination efficiency using a tRNA reporter gene orthogonal to the tRNA of an experimental strain. More specifically, the present invention relates to an operon sequence comprising n transcription termination sites, wherein the transcription termination at the i-th transcription termination site i (n is a positive integer, and i A method for measuring the in vivo transcription termination efficiency in (where n is a positive integer less than or equal to n), comprising: (A) preparing a tRNA reporter gene having orthogonality to the tRNA of the experimental strain; (B) the tRNA reporter gene (upstream of the promoter of the operon ~ transcription termination i The gene of the upstream) sequence up or (upstream of the promoter of the above operon ~ transcription termination i gene of the downstream) sequence down Plasmids fused respectively in a transcriptional manner up and plasmid down Step of producing; (C) the above plasmid upand plasmid down Recombinant strains transformed into each experimental strain up and recombinant strains down A step of producing; and (D) the recombinant strain up and recombinant strains down The present invention relates to a method for measuring transcription termination efficiency in vivo, comprising the step of quantifying the expression of each tRNA reporter gene in vivo.

[0011] The present invention relates to an operon sequence comprising n transcription termination sites, wherein the transcription termination at the i-th transcription termination site i (n is a positive integer, and i This invention relates to a method for measuring the in vivo transcription termination efficiency in a positive integer less than or equal to n. An operon consists of a series of structural genes transcribed by a common promoter. The present invention relates to transcription termination i Operon genes or transcription terminations including i It is characterized by measuring transcription termination efficiency in the in vivo expression of a tRNA reporter gene that is transcriptionally fused with an operon gene that does not contain .

[0012] Each step is explained in more detail below.

[0013] Step (A) above is the step of preparing a tRNA reporter gene.

[0014] In order to accurately measure the transcription of a tRNA reporter gene by an artificially introduced tRNA reporter gene within an experimental strain, endogenous factors of the experimental strain must not affect the transcription of the tRNA reporter gene. Therefore, in this step, a tRNA reporter gene that is orthogonal to the tRNA of the experimental strain is prepared. As a result, the corresponding tRNA reporter gene is not expressed in the strain itself, and since the expressed tRNA reporter gene does not react with the intrinsic molecules of the experimental strain, the number of tRNA reporter genes transcribed by the plasmid introduced in the subsequent step can be directly measured.

[0015] (B) Step is the transfer completion to measure transfer efficiency. i The step involves constructing a plasmid by transcriptionally fusing an operon-derived gene, with or without the tRNA reporter gene, with the tRNA reporter gene. By transcriptional fusion, the transcription of the tRNA reporter gene is regulated by the operon-derived gene. To this end, the operon-derived gene must include at least a promoter region. Optionally, it may include a regulatory gene region of the operon upstream of the promoter. Therefore, the above operon-derived gene starts upstream of the promoter, and transcription termination within the operon-derived gene i Includes sequences upstream or downstream of the gene up and genes down It is desirable that the upstream position of the operon promoter, which is the starting position, is the same. The above transcription termination i It may be an exact location as in the example, or it may be a region known to have a termination location. Accordingly, each of the above operon genes is a gene up and genes down It is referred to as a plasmid, wherein each of the above genes and the tRNA reporter gene are transcriptionally fused. up and plasmiddown It is called [this].

[0016] Transcription of the tRNA reporter gene in the above plasmid is controlled by a promoter included in the above operon gene.

[0017] Step (C) is the plasmid produced in Step (B) above. up or plasmid down Recombinant strain produced by transforming the experimental strain up and recombinant strains down This is the stage of constructing. By introducing a plasmid, the structural genes of the operon can be expressed in the recombinant strain.

[0018] (D) Step is the recombinant strain up and recombinant strains down This is a step of quantifying the expression of each in vivo tRNA reporter gene.

[0019] Recombinant strain up Silver Warrior Termination i Since it does not contain, transcription terminates when expression begins. i Transcription does not terminate there and continues to the tRNA reporter gene. On the other hand, recombinant strain down Silver Warrior Termination i Since it includes, transcription terminates when operon gene expression is initiated. i In some cases, transcription terminates and the tRNA reporter gene is not expressed, while in others, transcription continues and the tRNA reporter gene is expressed. Therefore, recombinant strain up and recombinant strains down Transcription termination by quantifying and comparing the expression of in vivo tRNA reporter genes, respectively i You can check the warrior termination efficiency in. Warrior Termination i Termination efficiency representing the transaction termination efficiency in i It can be analyzed qualitatively, but it can also be calculated quantitatively using the following formula.

[0020]

[0021] Quantification of the above tRNA can be performed by qRT-PCR, Northern blotting, β-galactosidase analysis, or primer expansion analysis. Among these, qRT-PCR is more advantageous because it can detect small changes in the expression level of tRNA reporter genes, allows for rapid analysis, and does not require exogenous substrates.

[0022] In particular, the method of the present invention is a transcription termination in which transcription is terminated by Rho-dependent termination. i It can be usefully applied to measure termination efficiency in [the context]. In the case of Rho-dependent termination, since transcripts are rapidly degraded, it is impossible to measure termination efficiency by transcriptome analysis; therefore, conventionally, transcription termination efficiency was indirectly measured by measuring the expression intensity of proteins. However, due to differences in translation rates, the amount of transcription and the amount of translation do not necessarily show a linear relationship. According to the method of the present invention, since transcription efficiency is directly measured by the amount of tRNA reporter gene representing the number of transcribed transcripts, bias inherent in translation can be excluded.

[0023] The expression level of the tRNA reporter gene measured in step (D) above depends on the synthesis rate and degradation rate of the corresponding gene. Therefore, in order to verify whether the measured transcription efficiency reflects the synthesis rate, i.e., the amount of transcription, the half-life of the tRNA reporter gene expressed in each recombinant strain can be further analyzed. Recombinant strain up and recombinant strains down If the half-lives measured in the above strains are identical within the margin of error, the difference in the expression amount of the tRNA reporter gene between the strains depends entirely on the difference in the amount of transcription, and thus the measured transcription efficiency can be verified.

[0024] In addition, steps (D) and (E) above can be performed on a mutant strain lacking Rho function relative to the experimental strain above to further measure the expression of the in vivo tRNA reporter gene. According to the above measurement, transcription termination i In this, it is possible to determine whether transcription termination is Rho-dependent and to validate the measured termination efficiency values. When transcription is terminated by Rho-independent termination, the termination efficiency in strains with Rho function i Termination efficiency in mutant strains lacking Rho function i ...will show similar values. On the other hand, if transcription is terminated by Rho-dependent termination, the measured transcription termination efficiency will be within the margin of error of 0 because Rho cannot function in mutant strains and therefore cannot induce transcription termination.

[0025] In the embodiments of the present invention E. coli Using as the experimental strain, and with the tRNA reporter gene E. coli tRNA that is orthogonal to the tRNA arg Using, three Rho-dependent locations and one Rho-independent location are known gal The usefulness of the present invention was confirmed by calculating the termination efficiency for each termination position of the operon. However, since this is intended to confirm that the method of the present invention can be effectively applied as a specific example, a person skilled in the art would find it easy to design an experiment by expanding any operon, experimental strain, and tRNA reporter gene by taking into account the description of the embodiments of the present invention, and to measure the transcription termination efficiency through this.

[0026] Another application of the present invention relates to a method for identifying Rho-dependent termination sites in vivo within an operon sequence containing Rho-dependent termination sites. While Nadira et al. and Di Salvo et al. constructed models to predict Rho-dependent termination sites, Chhakchhuak et al. confirmed through experiments that there is a need to improve the accuracy of the models. Therefore, an experimental method capable of identifying Rho-dependent termination sites is required. However, there are still various problems regarding the in vivo identification of Rho-dependent termination sites.

[0027] Accordingly, the Rho-dependent termination site can be identified in vivo by partially modifying the above-mentioned method for measuring transcription termination efficiency. The above-mentioned method comprises, in detail: (A) a step of preparing a tRNA reporter gene having orthogonality with the tRNA of an experimental strain; (B) a step of constructing a gene library consisting of a sequence from the promoter of the operon to any location upstream or downstream of the expected region of the Rho-dependent termination site; (C) a step of constructing a plasmid library in which each gene in the gene library and the tRNA reporter gene are transcriptionally fused; (D) a step of constructing a library of recombinant strains in which each plasmid in the plasmid library is transformed into an experimental strain; and (E) a step of quantifying the expression of the tRNA reporter gene in vivo of each strain.

[0028] In describing the embodiments of the present invention, the detailed description of each step applies to the content described in the method for measuring transfer efficiency. However, in the method for measuring transfer termination efficiency, transfer termination to a specific location or area i In contrast to knowing , this method involves transcription termination i It is assumed that only the predicted region is known, but the exact location is unknown. Therefore, a gene library is constructed to transcribe and fuse the tRNA reporter gene; in this case, the genes constituting the library each consist of sequences extending from the upstream of the operon's promoter to an arbitrary location upstream or downstream of the predicted region of the Rho-dependent termination site. After transcribing and fusing the tRNA reporter gene with each gene constituting the gene library, the resulting structure is transformed into an experimental strain to construct a library of recombinant strains, and the expression of the tRNA reporter gene in each strain is quantified. If the expression of the tRNA reporter gene in vivo is identical to that of the recombinant strain using the gene up to (the upstream of the operon's promoter to the upstream of the predicted region of the Rho-dependent termination site), then the gene used for the transformation of the recombinant strain is transcriptionally terminated. i It can be confirmed that it does not include. On the other hand, if the expression of the tRNA reporter gene has decreased, the gene used for the transformation of the corresponding recombinant strain has transcription termination. i It includes. Therefore, the Rho-dependent termination site can be identified by the expression level of the tRNA reporter gene.

[0029] Similar to the method for confirming transcription termination efficiency, the corresponding transcription termination is further measured by performing steps (D) and (E) above using a mutant strain lacking Rho function to additionally measure the expression of the in vivo tRNA reporter gene. i It can be confirmed that this is due to Rho-dependent termination. Effects of the invention

[0030] As described above, the present invention solves the problems of the prior art, allowing the transcription efficiency of transcripts due to Rho-dependent termination in vivo to be measured directly and quantitatively, which can be usefully utilized for regulating gene expression.

[0031] In addition, according to the present invention, the problem of the prior art, in which it was difficult to confirm the exact termination location due to the instability of the transcriptome, is resolved, thereby enabling the confirmation of the Rho-dependent termination location in vivo. Brief explanation of the drawing

[0032] Figure 1 is a cleavage map of a plasmid showing the structure of a plasmid prepared by the present method. Figure 2 is gal E- gal This is a graph showing the efficiency of Rho-dependent termination at the T-cistron junction. Fig. 3 is gal T- gal This is a graph showing the efficiency of Rho-dependent termination at the K-cistron junction. Fig. 4 is gal This is a graph showing the efficiency of RIT and RDT at the operon end. Specific details for implementing the invention

[0033] The present invention will be described in more detail below with reference to the attached drawings and embodiments. However, these drawings and embodiments are merely examples to easily explain the content and scope of the technical concept of the present invention, and the technical scope of the present invention is not limited or altered by them. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical concept of the present invention based on these examples.

[0034] [Example]

[0035] I. Materials and Methods

[0036] 1) Culture of the strain

[0037] In the following examples E. coli MG1655, HME60( rho-15) and MG1655 Δspf A strain was used. E. coliChromosome deletion strains of MG1655 were generated using phage lambda red-mediated recombination according to the method of Datsenko KA and Wanner BL (Proc Natl Acad Sci USA 2000 97:6640-6645). The DH5α strain was used for plasmid construction, and the primers used in this invention are summarized in Table 1 below.

[0038]

[0039] All cells were tested for optical density (OD) at 600 nm in Lysogeny Broth supplemented with 0.5% (w / v) galactose and chloramphenicol (15 µg / ml) or ampicillin (100 µg / ml) at 37°C. 600 Cultured until it reached an exponential growth stage where ) was 0.6. Subsequently, RNA isolation was performed.

[0040] 2) Design and Construction of Plasmids

[0041] B. albidum tRNA from arg The araX sequence corresponding to is the tRNA listed in Table 1 arg -F and tRNA arg -R primer pairs were used for amplification by PCR. The amplified PCR fragment was cleaved with Hind III, and the chloramphenicol resistance gene (which lacks the promoter of pKK232-8 (GE Healthcare, USA), an intermediate copy number plasmid derived from pBR322) cat The pHL1141 plasmid was constructed by inserting it upstream of ). To analyze Rho-dependent termination, gal Operon DNA sequence in pHL1141 arg Located upstream of X Bam HI and Sal I cloned between restriction enzyme sites.

[0042] Specifically, to generate plasmids pHL1031, pHL1193, pHL2000, pHL2184, pHL4270, pHL4344, and pHL4444, PCR amplification was performed using gal+70-F (SEQ No. 5) and primer pairs of SEQ Nos. 6–12, respectively. gal of coordinates -73~1,031, -73~1,193, -73~2,000, -73~2,184, -73~4,270, -73~4,333 and -73~4,444 gal The operon DNA sequence was obtained. The generated PCR fragment is Bam HI and Sal It was ligated into pHL1141 cleaved with I. The structure of the plasmid was confirmed by DNA sequencing.

[0043] Figure 1 shows the structure of the plasmids prepared by the present method based on the plasmid cleavage map. Each plasmid is described once again with reference to Figure 1. In Figure 1, the yellow ori indicates the origin of plasmid replication, and is shown in green. cat and bla Each represents the chloramphenicol acetyltransferase and β-lactamase antibiotic resistance genes. rrnBT1 / T2 Transcription terminators are denoted as "T", and restriction enzymes Hind III, Sal I and Bam HI was shown. Plasmid pKK232-8 cat tRNA upstream of the gene arg pH1141 was generated by inserting X (blue). pH1141 tRNA arg In front of genes gal pHL1031, pHL1193, pHL2000, pHL2184, pHL4270, pHL4344, and pHL4444 were constructed by cloning the DNA fragments of the operon. gal The Rho-dependent termination site of the DNA fragment galThe end of the cistron is marked with a T.

[0044] 3) RNA preparation and reverse transcription quantitative PCR (qRT-PCR)

[0045] Using the Direct-zol RNA MiniPrep Kit (Zymo Research, USA) according to the manufacturer's instructions, purified cell lysate (2×10⁶ 8 Total RNA was purified from cells. 1 µg of total RNA was reacted with Turbo DNase I (Thermo Fisher Scientific, USA) to remove genomic or plasmid DNA, and then reverse transcribed with a reaction volume of 20 µl according to the method described in J Bacteriol 2014 196:2598-2606. The results for each sample were compared with an internal control encoding 16S rRNA. rrsB Normalized using rrsB ...was amplified using the primer pair of SEQ ID NO. 1 and SEQ ID NO. 2 in Table 1. The qRT-PCR reaction was repeated three times, and three independent experiments were additionally analyzed using the mean Ct values. Expression in each sample was expressed as mean ± SD. One-way analysis of variance (ANOVA) on ΔΔCT values ​​was performed using the Bonferroni test. GraphPad Prism 9.0 was used for ANOVA and graphing.

[0046] 4) RNA stability analysis

[0047] Each plasmid Using the available wild-type (WT) MG1655 cells galE , tRNA arg , 16S rRNA, cat The mRNA decay rate was investigated. To confirm RNA stability, 100 µg / mL of the transcription inhibitor rifampicin was administered at an OD level. 600It was added to LB cultured cells with a value of 0.6. Samples were collected at 0, 2, 4, and 8 minutes after the addition of rifampicin, and total RNA was extracted for qRT-PCR analysis. 16S rRNA was used to standardize the results. Relative expression was calculated by averaging the change in scale of three copies across three independent experiments using the 2-ΔΔCT method.

[0048] II. Results

[0049] 1) Establishment of an In Vivo Rho-Dependent Termination Detection System

[0050] B. albidum tRNA arg A 77 bp gene encoding arg By qRT-PCR using a pBR322-derived plasmid containing X gal Rho-dependent termination in the operon was analyzed. gal The operon is gal E- gal T- gal K- gal It includes the structural gene of M, gal E- gal T and gal T- gal K-sistron junction and gal The Rho-dependent termination location of M downstream is known. B. albidum tRNA derived arg The reporter E. coli It is known that it is orthogonal to tRNA and is quite stable, so it can be detected by qRT-PCR analysis.

[0051] First, tRNA arg As a result of testing the orthogonality of, arg In MG1655-pKK232 that does not contain the X gene, tRNA arg tRNA was not detected arg Is E. coli Orthogonality to tRNA was confirmed (data not shown). Subsequently, in the MG-1655-pHL1141 strain, tRNA argThe half-life of was measured by qRT-PCR. Contrary to expectations, tRNA arg It did not exhibit strong stability, and instead, as stated in Table 2 gal It exhibited a half-life similar to mRNA. cat It exhibited a half-life of about 1.5 minutes, and 16S rRNA was very stable with no degradation observed.

[0052]

[0053] various gal Seven plasmids (Fig. 1) formed by fusing the operon sequences, respectively E. coli Transformed into the MG1655 strain, and tRNA expressed in the transformed strain arg The half-life of was measured. As can be seen in Table 3, tRNA arg The half-life was similar, ranging from 1.0 to 1.3 minutes. The amount of RNA quantified by qRT-PCR depends on the RNA synthesis rate and the RNA degradation rate. tRNA in the above plasmids arg Since it was confirmed that it degrades at a similar rate, tRNA in the above-mentioned transformed strains arg The amount of before and after the estimated Rho-dependent termination position gal Initiated by the promoter gal It can be seen as representing the amount of transcripts.

[0054]

[0055] 2) Rho-dependent termination at the galE-galT cistron junction

[0056] In vivo, before and after Rho-dependent termination (RDT) at position 1,183 gal Rho-dependent termination efficiency was evaluated by measuring the amount of tRNA. To measure transcription prior to RDT gal The operon region is -73 to 1,031, and for measuring transcription after RDT gal The operon regions are -73 to 1,193, and the corresponding plasmids are pHL1031 and pHL1193, respectively. ga The corresponding area of ​​l is gal It includes all cis-acting elements essential for expression. tRNA derived from pHL1031 and pHL1193 arg The amount is 1,183 RDT before and after gal Initiated by the promoter gal Indicates the amount of transcripts. tRNA in two strains harboring pHL1031 and pHL1193 plasmids, respectively. arg The expression of was measured by qRT-PCR, and the results are shown in Fig. 2. In Fig. 2A, tRNA in the MG1655-pHL1031 strain arg tRNA expression in the MG1655-pHL1193 strain arg The expression of was 64±19% (0.001 <p<0.01). 이는 MG1655의 1,183 위치에서 Rho로 인한 전사 종결이 약 36% 발생함을 시사한다. 반면 도 2의 B에 도시된 바와 같이 Rho가 기능하지 않는 HME60( rho ::Amp R tRNA in cells arg The expression of did not differ significantly from that of MG1655-pHL1031 (p>0.05), galIE-gal tRNA at the T junction arg It was confirmed that the expression level of reflects Rho-dependent termination. Table 4 shows the transcription termination efficiency at each position.

[0057]

[0058] 3) Rho-dependent termination at the galT-galK cistron junction

[0059] Using strains transformed with pHL2000 and pHL2184 plasmids, respectively, at locations 2121–2125 gal ET- shortThe efficiency of the RDT known to generate was measured. Since cell-free assays demonstrated that Rho terminates transcription at 2,184, the tRNA at MG1655-pHL2184 arg It can be expected that the expression of will be lower than in MG1655-pHL2000. Indeed, in Fig. 3A, tRNA in MG1655-pHL2184 arg The expression of tRNA in MG1655-pHL2000 arg It was indicated that 26% of transcription was terminated by Rho at the corresponding RDT site with 74±19% of the expression.

[0060] Spot42, a non-coding sRNA with a length of 109 nucleotides, ga lT- gal It is known to enhance RDT by binding to the middle of multiple cistron mRNA at the K-cistron junction. As shown in Fig. 3B, Δ, in which the gene encoding Spot42 is deleted SPF tRNA in the -pHL2184 strain arg Expression is Δ SPF With 92±10% in -pHL2000, it demonstrates that in the absence of Spot42, it is difficult for Rho to terminate the transcription even if an RDT site exists. This indicates that Spot42 ga This implies that it is an important factor inducing RDTs that generate lET mRNA. Additionally, Figure 3C shows tRNA in HME60-pHL2184 and HME60-2000. arg Showing that expression is nearly identical, tRNA in MG1655-pHL2000 and MG1655-pHL2184, as in Fig. 2B. arg It was indicated that the expression of reflects Rho-dependent termination.

[0061] 4) RDT and RIT at the operon terminal

[0062] galAt the operon terminus, transcription is terminated by the upstream RIT and downstream RDT. At 4,409, transcription is terminated by Rho and subsequently rapidly degraded by exonucleases. The known RIT and RDT sites are 4,315 and 4,409, respectively, and tRNA in MG1655 strains transformed with the pHL4270 (RIT-pre-RDT), pHL4344 (post-RIT-pre-RDT), and pHL4444 (RIT-post-RDT) plasmids, respectively. arg Termination efficiency was evaluated by measuring expression using qRT-PCR. As indicated in Table 3, the half-lives of tRNA were nearly similar in each strain, so tRNA in each strain arg Transcription termination efficiency can be evaluated by measuring expression levels.

[0063] Figure 4 is a graph showing the results, and in Figure 4A, the tRNA of MG1655-pHL4344 against MG1655-pHL4270 arg The expression level is 67±2%, showing an RIT efficiency of 33%. tRNA in MG1655-pHL4444 for MG1655-pHL4720 arg With an expression level of 25±8%, it can be calculated that the total efficiency of transcription terminated by RIT and RDT at the operon terminus is 75%, and the RDT efficiency is 63%. These results are also listed in Table 4. Figure 4B shows HME60( rho ::Amp R It shows the efficiency of RIT and RDT in ) cells. Specifically, tRNA in HME60-pHL4344 against HME60-pHL4270. arg The expression level is 68±2%, and therefore the RIT efficiency is 32%. This value is nearly identical to the RIT efficiency measured in MG1655, confirming that it was hardly affected by the presence or absence of Rho function. tRNA in HME60-pHL4444 for HME60-pHL4720 argThe expression level was 90±14%, and the total efficiency of transcription terminated by RIT and RDT at the operon terminus dropped to 10%, but was not significant compared to HME60-pHL4270. This suggests that the termination efficiency at the operon terminus corresponds to the RDT efficiency.

Claims

Claim 1 In an operon sequence containing n transcription termination sites, the transcription termination at the i-th transcription termination site i (n is a positive integer, and i A method for measuring the in vivo transcription termination efficiency in (where n is a positive integer less than or equal to n), comprising: (A) a step of preparing a tRNA reporter gene having orthogonality to the tRNA of an experimental strain; (B) the tRNA reporter gene (upstream of the promoter of the operon ~ transcription termination i The gene of the upstream) sequence up or (upstream of the promoter of the above operon ~ transcription termination i gene of the downstream) sequence down Plasmids fused respectively in a transcriptional manner up and plasmid down Step of producing; (C) the above plasmid up and plasmid down Recombinant strains transformed into each experimental strain up and recombinant strains down A step of producing; and (D) the recombinant strain up and recombinant strains down A method for measuring in vivo Rho-dependent transcription termination efficiency, comprising the step of quantifying the expression of each in vivo tRNA reporter gene. Claim 2 In claim 1, the transfer termination i A method for measuring in vivo Rho-dependent transcription termination efficiency, characterized in that the transcription termination efficiency in [location] is calculated by the following formula. Claim 3 In claim 1, the transfer termination i A method for measuring in vivo Rho-dependent transcription termination efficiency characterized by being a Rho-dependent transcription termination site. Claim 4 A method for measuring in vivo Rho-dependent transcription termination efficiency, characterized in that, in any one of claims 1 to 3, the quantification of the tRNA is performed by qRT-PCR, Northern blotting, β-galactosidase analysis, or primer expansion analysis. Claim 5 A method for measuring in vivo Rho-dependent transcription termination efficiency, characterized in that, in any one of claims 1 to 3, the half-life of a tRNA reporter gene expressed in each recombinant strain is further analyzed. Claim 6 A method for measuring in vivo Rho-dependent transcription termination efficiency, characterized in that, in any one of claims 1 to 3, steps (D) and (E) are performed using a mutant strain lacking Rho function to further measure the expression of an in vivo tRNA reporter gene. Claim 7 delete Claim 8 delete

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