• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권3호
• /
• pp.258-261
• /
• 2006

The addition of zeolite particles enhances the stability of mRNA molecules in a cell-free protein synthesis system. When $20{\mu}g/{\mu}L$ of zeolite (Y5.4) is added to a reaction mixture of cell-free protein synthesis, a substantial increase in protein synthesis is observed. The stabilizing effect of zeolite is most dearly observed in an in vitro translation reaction directed by purified mRNA, as opposed to a coupled transcription and translation reaction. Upon the addition of zeolite in the in vitro translation reaction, the life span of the mRNA molecules is substantially extended, leading to an 80% increase in protein synthesis. The effect of zeolite upon the mRNA stability appears be strongly related to the cation exchange (potassium to sodium) reaction. Our results demonstrate the possibility of modifying this biological process using heterogeneous, non-biological substances in a cell-free protein synthesis system.

• Journal of Microbiology and Biotechnology
• /
• 제16권3호
• /
• pp.355-359
• /
• 2006

The functional stability of mRNA is one of the crucial factors affecting the efficiency of in vitro translation. As the rapid degradation of mRNA in the cell extract (S30 extract) causes early termination of the translational reactions, extending the mRNA half-life will improve the productivity of the in vitro protein synthesis. Thus, a simple PCR-based method is introduced to increase the stability of mRNA in an S30 extract. The target genes are PCR-amplified with primers designed to make the ends of the transcribed mRNA molecule anneal to each other. When compared with normal mRNA, the mRNA with the annealing sequences resulted in an approximately 2-fold increase of protein synthesis in an in vitro translation reaction. In addition, sequential transcription and translation reactions in a single tube enabled direct protein expression from the PCR-amplified genes without any separate purification of the mRNA.

• Asian-Australasian Journal of Animal Sciences
• /
• 제9권2호
• /
• pp.171-174
• /
• 1996

Muscle protein synthesis in vitro was measured in chicks fed low-protein(10% CP) and control(20% CP) diets. Right leg muscles (M. gastrocnemius) were mounted on a support made of stainless steel to stretch in constant tension, whereas left leg muscles were unmounted. Both leg muscles were incubated in Dulbecco's modified Eagle's medium including L-[$4-^3H$] phenylalanine for 60 min to measure in vitro protein synthesis. There was no significant difference in fractional synthesis rate(FSR) of muscle protein between both dietary protein levels, whereas FSR with stretch in constant tension was significantly higher than that without constant tension due to an increase in the absolute synthesis rate(ASR) per unit RNA(the efficiency of RNA to synthesize protein). The ASR of muscle protein in chicks fed the control diet was significantly higher than that in the low-protein diet group.

• 생명과학회지
• /
• 제15권5호
• /
• pp.755-762
• /
• 2005

세포 내에서 매우 적은 양으로 존재하는 RNA 전사체와 기능적으로 동일하거나 비슷한 RNA를 RNA 중합효소를 써서 in vitro에서 생화학적으로 의미 있는 양 만큼을 합성할 수 있다. T7 RNA중합효소를 발현하는 재조합 유전자를 지닌 대장균주 BL21/pAR1219로부터 순수한 T7 RNA중합효소를 손쉽게 얻는 방법을 본 논문에서 소개한다. 황산암모늄 분획화와 sephadex SP 컬럼 크로마토그래피법으로써 여타의 방법과 비교하여 더 간단하고 빠르게, 그리고 경제적으로 T7 RNA 중합효소를 분리할 수 있었다. 정제된 T7 RNA중합효소를 이용하여 보통의 화학적 합성법으로 불가능한 긴 길이(1.54 kb)의 RNA전사체를 합성 하였다. 한편,정제된 T7 RNA중합효소에 의해 생성된 망치머리 리보자임은 표적 RNA를 in vitro에서 절단함으로써, 생성된 RNA가 생화학적 기능성을 유지한다는 것을 입증하였다 따라서 본 연구에서 소개되는 절차들은 다양한 길이의 RNA를 목적에 따라 간단하고 경제적으로 합성하는데 유용하게 이용될 수 있다.

• Bulletin of the Korean Chemical Society
• /
• 제32권10호
• /
• pp.3779-3782
• /
• 2011

• 미생물과산업
• /
• 제14권1호
• /
• pp.7-11
• /
• 1988

Efficient in vitro RNA synthesis can be easily accomplished from cloned DNA using bactrio-phage SP6, T7 or T3 RNA polymerase. Despite its popularity as in vitro transcription system, molecular mechanisms of bacteriophage transcription has not been studied, although physical and catalytic properties of several phage RNA polymerases have well been documented (1). Only recently the T7 promoter has been physically mapped by footprinting of the T7 RNA polymerase (2,3). These simple phage systems, however, could be useful for detailed molecular studies of transcription.

• BMB Reports
• /
• 제43권11호
• /
• pp.732-737
• /
• 2010

RNA interference is a post-transcriptional silencing mechanism triggered by the bioavailability and/or exogenous introduction of double-stranded RNA (dsRNA) into cells. Here we describe a novel method for the synthesis of siRNA in a single vessel. The method employs in vitro transcription and a single-stranded DNA (ssDNA) template and design, which incorporates upon self-annealing, two promoters, two templates, and three loop regions. Using this method of synthesis we generated efficacious siRNAs designed to silence both exogenous and endogenous genes in mammalian cells. Due to its unique design the single-stranded template is easily amenable to adaptation for attachment to surface platforms for synthesis of siRNAs. A siRNA synthesis platform was generated using a 3' end-biotinylated ssDNA template tethered to a streptavidin coated surface that generates stable siRNAs under multiple cycles of production. Together these data demonstrate a unique and robust method for scalable siRNA synthesis with potential application in RNAi-based array systems.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제11권5호
• /
• pp.420-424
• /
• 2006

A simple method for depleting E. coliS30 extracts of endogenous tRNA has been developed. An $ethanolamine-Sepharose^{(R)}$ column equilibrated with water selectively captured the tRNA molecules in E. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful for in vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.

• Molecules and Cells
• /
• 제19권2호
• /
• pp.239-245
• /
• 2005

Factor for inversion stimulation (FIS), the Escherichia coli protein, is a positive regulator of the transcription of genes that encode stable RNA species, such as rRNA and tRNA. Transcription of the rnpB gene encoding M1 RNA, the catalytic subunit of E. coli RNase P, rapidly declines under stringent conditions, as does that of other stable RNAs. There are multiple putative FIS binding sites upstream of the rnpB promoter. We tested whether FIS binds to these sites, and if so, how it affects rnpB transcription. In vitro binding assays revealed specific binding of FIS to multiple sites in the rnpB promoter region. Interestingly, FIS bound not only to the upstream region of the promoter, but also to the region from +4 to +18. FIS activated rnpB transcription in vitro, but the level of activation was much lower than that of the rrnB promoter for rRNA. We also examined the effects of FIS on rnpB transcription in vivo using isogenic $fis^+$ and $fis^-$ strains. rnpB transcription was higher in the $fis^-$ than the $fis^+$ cells during the transitions from lag to exponential phase, and from exponential to stationary phase.

• Bulletin of the Korean Chemical Society
• /
• 제29권6호
• /
• pp.1137-1140
• /
• 2008

M1 RNA, the catalytic subunit of Escherichia coli RNase P, is an essential ribozyme that processes the 5' leader sequence of tRNA precursors (ptRNAs). Using KS2003, an E. coli strain generating only low levels of M1 RNA, which showed growth defects, we examined whether M1 RNA is involved in polycistronic mRNA processing or degradation. Microarray analysis of total RNA from KS2003 revealed six polycistronic operon mRNAs (acpP-fabF, cysDNC, flgAMN, lepAB, phoPQ, and puuCBE) showing large differences in expression between the adjacent genes in the same mRNA transcript compared with the KS2001 wild type strain. Model substrates spanning an adjacent pair of genes for each polycistronic mRNA were tested for RNase P cleavage in vitro. Five model RNAs (cysNC, flgMN, lepAB, phoPQ, and puuBE) were cleaved by RNase P holoenzyme but not by M1 RNA alone. However, the cleavages occurred at non-ptRNA-like cleavage sites, with much less efficiency than the cleavage of ptRNA. Since cleavage products generated by RNase P from a polycistronic mRNA can have different in vivo stabilities, our results suggest that RNase P cleavage may lead to differential expression of each cistron.