Effect of Transcription Terminators on Expression of Human Lipocortin-1 in Recombinant Saccharomyces cerevisiae

Kim, Byung Moon,Nam, Soo Wan,Chung, Bong Hyun,Park, Young Hoon,Rhee, Sang Ki 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.4

The vector systems for the expression and secretion of human lipocortin-1 (LC1) from Saccharomyces cerevisiae were constructed with GAL10 promoter and the prepro leader sequence of mating factor-αl. They were further constructed to contain three different transcription terminators; GAL7 terminator, LC1 terminator and a fused form of these two terminators. The expression and secretion levels of LC1 were compared to investigate the effect of transcription terminators on the LC1 gene expression. For the expression cassettes employing the GAL7 terminator or the terminator of fused form, the expression levels of LC1 were measured by scanning the immunoreactive LC1 protein bands, and were found to be 0.27g/ℓ and 0.32g/ℓ, respectively. The highest expression level of 0.54g/ℓ was obtained with the expression vector containing the LC1 transcription terminator. In all expression cassettes, the majority of LC1 proteins expressed were retained intracellularly, indicating a low secretion efficiency of about 5%. The high expression level of LC1 was explained by the great content and stability of LC1 mRNA transcribed from the LC1 terminator-employing vector. The results of this study demonstrate that the LC1 transcription terminator functions for the expression of LC1 in S. cerevisiae better than the GAL7 terminator.

Rho-dependent Transcription Termination: More Questions than Answers

Ranjan Sen,Jisha Chalissery,Sharmistha Banerjee,Irfan Bandey 한국미생물학회 2006 The journal of microbiology Vol.44 No.1

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize ‘the knowns’ and ‘the unknowns’ of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Rho-dependent Transcription Termination: More Questions than Answers

Banerjee Sharmistha,Chalissery Jisha,Bandey Irfan,Sen Ranjan The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.1

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize 'the knowns' and 'the unknowns' of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Activity analysis of LTR12C as an effective regulatory element of the RAE1 gene

Jung, Y.D.,Lee, H.E.,Jo, A.,Hiroo, I.,Cha, H.J.,Kim, H.S. Elsevier/North-Holland 2017 Gene Vol.634 No.-

Ribonucleic acid export 1 (RAE1) plays an important role in the export of mature mRNAs from the nucleus to the cytoplasm. Long terminal repeats (LTRs) became integrated into the human genome during primate evolution. One such repeat element, LTR12C, lies within a predicted regulatory region located upstream of the RAE1 gene. We examined the transcriptional activity of LTR12C by using the luciferase assay, and showed that the tandem repeat region (TRR) located within LTR12C was required for its regulatory function. A bioinformatics analysis revealed that the LTR12C element had multiple transcription factor binding sites specific for nuclear transcription factor Y (NF-Y), and the promoter activity of LTR12C was significantly decreased after NF-Y knockdown. Additionally, we discovered novel data indicating that LTR12C was initially inserted into the gorilla genome. Taken together, our results reveal that the TRR of LTR12C has powerful regulatory activity due to its NF-Y binding sites, and the integration of the LTR12C element into the primate genome during evolution may have affected RAE1 transcription.

Alu Tandem Sequences Inhibit GFP Gene Expression by Triggering Chromatin Wrapping

Xiu Fang Wang,Xiao Yan Wang,Jing Liu,Jing Jing Feng,Wen Li Mu,Xiao Juan Shi,Qin Qing Yang,Xiao Cui Duan,Ying Xie,Zhan Jun Lu 한국유전학회 2009 Genes & Genomics Vol.31 No.3

Alu elements belonging to the short interspersed nuclear elements (SINE) of repetitive elements are present in more than one million copies which altogether represent 10% of the whole human genome. In this study, the roles of Alu tandem sequences in the process of GFP gene (GFP) expression and packing into chromatin of its DNA were studied. To detect the effect of Alu repeats on gene expression, different copies of Alus were inserted GFP downstream respectively in pEGFP-C1 vector. We found that Alu sequences decreased the amount of GFP transcription, the percentage of GFP positive cells and the accessibility to DNase I in length-dependent manner. Inserting Alu caused the production of higher-molecular-mass RNA, indicating Alu sequence did not induce premature transcriptional termination. Tight packing chromatins keep silent and resist to DNase I digestion, which is a general phenomenon. We suggested that head and tail tandem Alu sequences suppressed GFP expression in length dependent manner by triggering chromatin packing.

Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes

Wang, Xun,N, Monford Paul Abishek,Jeon, Heung Jin,Lee, Yonho,He, Jin,Adhya, Sankar,Lim, Heon M. National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.10

<P><B>Significance</B></P><P>Transcription termination by RNA polymerase in prokaryotes is well understood in contrast to similar mechanisms in higher organisms. Despite the in vitro occurrence of two types of demonstrable transcription termination events in prokaryotes at the end of transcription units, they are obscured in vivo in two ways: suppression of termination by traversing of the RNA polymerase through the termination sites when coupled to translation, or by further processing of the actual terminated RNA 3′ ends by RNases, as in eukaryotes.</P><P>Two kinds of signal-dependent transcription termination and RNA release mechanisms have been established in prokaryotes in vitro by: (<I>i</I>) binding of Rho to cytidine-rich nascent RNA [Rho-dependent termination (RDT)], and (<I>ii</I>) the formation of a hairpin structure in the nascent RNA, ending predominantly with uridine residues [Rho-independent termination (RIT)]. As shown here, the two signals act independently of each other and can be regulated (suppressed) by translation–transcription coupling in vivo. When not suppressed, both RIT- and RDT-mediated transcription termination do occur, but ribonucleolytic processing generates defined new 3′ ends in the terminated RNA molecules. The actual termination events at the end of transcription units are masked by generation of new processed 3′ RNA ends; thus the in vivo 3′ ends do not define termination sites. We predict generation of 3′ ends of mRNA by processing is a common phenomenon in prokaryotes as is the case in eukaryotes.</P>

DNA Light-strand Preferential Recognition of Human Mitochondria Transcription Termination Factor mTERF

Nam, Sang-Chul,Kang, Chang-Won Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.6

Transcription termination of the human mitochondrial genome requires specific binding to termination factor mTERF. In this study, mTERF was produced in E. coli and purified by two-step chromatography. mTERF-binding DNA sequences were isolated from a pool of randomized sequences by the repeated selection of bound sequences by gel-mobility shift assay and polymerase chain reaction. Sequencing and comparison of the 23 isolated clones revealed a 16-bp consensus sequence of 5'-GTG$\b{TGGC}$AGANCCNGG-3' in the light-strand (underlined residues were absolutely conserved), which nicely matched the genomic 13-bp terminator sequence 5'-$\b{TGGC}$AGAGCCCGG-3'. Moreover, mTERF binding assays of heteroduplex and single-stranded DNAs showed mTERF recognized the light strand in preference to the heavy strand. The preferential binding of mTERF with the light-strand may explain its distinct orientation-dependent termination activity.

Systematic Analysis on Transcription Unit Architecture of Streptomyces lividans TK24

이용재,이남일,정유진,황순규,김우리,조수형,( Bernhard O. Palsson ),조병관 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

Streptomyces lividans is an attractive host for heterologous production of proteins and secondary metabolites. In this study, the transcription unit (TU) architecture of S. lividans was elucidated by integrating four high-throughput data types, including dRNA-Seq, Term-Seq, RNA-Seq and Ribo-Seq. Total 1,300 TUs and the corresponding regulatory elements were elucidated from 1,978 transcription start sites and 1,640 transcript 3'-end positions. The TU information and regulatory elements identified will serve as invaluable resources for understanding the regulatory mechanisms of S. lividans and to elevate its industrial potential. ^** This work was supported by the Novo Nordisk Foundation (NNF10CC1016517). This work was also supported by the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957) and the Bio & Medical Technology Development Program (2018M3A9F3079664) through the National Research Foundation of Korea funded by the Ministry of Science and ICT.

Elucidating the Regulatory Elements for Transcription Termination and Posttranscriptional Processing in the Streptomyces clavuligerus Genome

Soonkyu HWANG,Namil LEE,Donghui CHOE,Yongjae LEE,Woori KIM,Yujin JEONG,Suhyung CHO,Bernhard PALSSON,Byung-Kwan CHO 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10

Identification of transcriptional regulatory elements in the GC-rich Streptomyces genome is essential for the production of novel biochemicals from secondary metabolite biosynthetic gene clusters. We identified the transcriptional regulatory elements in β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27064 by determining transcript 3¢-end positions (TEPs) using term-seq. Termination of transcription was governed by three classes of TEPs, of which each displayed unique sequence features. The data integration with transcriptome data generated transcription units (TUs) and transcription unit clusters (TUCs). TU architecture showed that the transcript abundance in TU isoforms of a TUC was potentially affected by the sequence context of their TEPs. We also identified TU features of XRE family regulator and DUF397 domain-containing protein, showing the abundance of bidirectional TEPs. Finally, we found potential cis- and trans-regulatory elements that played a major role in 5’ and 3’-UTR. These findings highlight the role of the regulatory elements in transcription termination and posttranscriptional processing in Streptomyces sp.

Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome

Kim, Kyumin,Heo, Dong-hyuk,Kim, Iktae,Suh, Jeong-Yong,Kim, Minkyu American Society for Biochemistry and Molecular Bi 2016 The Journal of biological chemistry Vol.291 No.25

<P>The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease.</P>