Efficient Transcription Termination Research Articles

Saccharomyces cerevisiae mRNA 3′ end forming signals are also involved in transcription termination

Previously, a 38-base-pair (bp) region in the 3' untranslated portion of the Saccharomyces cerevisiae iso-1-cytochrome c gene, was shown to be required for both normal CYC1 mRNA 3' end formation (Zaret and Sherman, 1982), and efficient transcription termination (Russo and Sherman, 1989). In another study, specific sequences such as TATATA, TACATA, and TAGTAGTA were shown to be involved in mRNA 3' end formation in S. cerevisiae (Russo et al., 1991). In this report, an in vivo plasmid stability assay has been utilized to show that these and related sequences are also involved in transcription termination, at varying efficiencies, and in an orientation-dependent manner. For example: the sequence TATATA appeared to terminate transcription almost as efficiently as the original wild type 38-bp region; whereas, the sequences TAGATATATGTAA and TACATA were less efficient, and TTTTTTTATA had little, if any, transcription termination function. In contrast, none of these sequences appeared to terminate transcription in the reverse orientation. Therefore, it appears that certain sequence signals capable of promoting mRNA 3' end formation in yeast, are also directly involved in transcription termination.

POSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli

Expression of the pro U operon of Escherichia coli is directly proportional to the osmolarity of the growth medium. The basal level of proU transcription is very low, but a large increase is triggered by a sudden rise in the external osmolarity. This increased expression is maintained for as long as the osmotic stimulus persists. We have capitalized upon these regulatory features of the proU operon and have constructed a series of expression vectors (pOSEX) permitting osmotically controlled expression of heterologous genes governed by regulatory signals of proU. The pOSEX vectors carry the pro U promoter, an upstream region required for high-level expression, and part of the first structural gene (proV), which acts as a silencer and is necessary to maintain low-level expression in low osmolarity media. An extended multiple cloning site (MCS) positioned at the 3' end of proV' permits the cloning of heterologous genes into the pOSEX plasmids, and efficient transcription terminators derived from the rrnB operon prevent deleterious read- through transcription into the vector portion. The properties of the pOSEX expression vectors were tested by positioning a promoterless lacZ (encoding β-galactosidase) gene from E. coli and the gcdA (encoding carboxytransferase) gene from the Gram+ bacterium Acidaminococcus fermentans under the control of the proU regulatory region. Efficient, osmoregulated and finely tuned expression of both lacZ and gcdA was achieved, and the amount of β-galactosidase and carboxytransferase synthesized were simply controlled by adjusting the osmolarity of the growth medium with various concentrations of NaCl.

Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series

A series of Escherichia coli-mycobacteria shuttle plasmids for the isolation and study of gene regulatory sequences was constructed. These pJEM vectors contain an efficient transcription terminator and multiple cloning sites and allow either operon or gene fusions to lacZ. By constructing operon fusions with pJEM15, we assessed various previously characterized mycobacterial promoters in the fast-growing species Mycobacterium smegmatis and the slow-growing species M. bovis BCG. Our results suggest that M. smegmatis and M. bovis BCG RNA polymerases do not share the same specificity. To isolate new mycobacterial promoters, an M. tuberculosis DNA library was generated, using pJEM13, and screened in M. smegmatis. Several Lac+ clones were isolated, and the beta-galactosidase activity was measured.

Production of pharmaceutical proteins from transgenic animals

Different systems are being studied and used to prepare recombinant proteins for pharmaceutical use. The blood, and still more the milk, from transgenic animals appear a very attractive source of pharmaceuticals. The cells from these animals are expected to produce well-matured proteins in potentially huge amounts. Several problems remain before this process becomes used in a large scale. Gene transfer remains a difficult and costly task for farm animals. The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency. Improvement of these vectors includes the choice of efficient promoters, introns and transcription terminators, the addition of matrix attached regions (MAR) and specialized chromatin sequences (SCS) to enhance the expression of the transgenes and to insulate them from the chromatin environment. Mice are routinely used to evaluate the gene constructs to be transferred into larger animals. Mice can also be utilized to prepare amounts as high as a few hundred mg of recombinant proteins from their milk. Rabbit appears adequate for amounts not higher than 1 kg per year. For larger quantities, goat, sheep, pig and cow are required. No recombinant proteins extracted from the blood or milk of transgenic animals are yet on the market. The relatively slow but real progress to improving the efficiency of this process inclines to be reasonably optimistic. Predictive reports suggest that 10% of the recombinant proteins, corresponding to a 100 million dollars annual market, will be prepared from the milk of transgenic animals by the end of the century.

Polyadenylation and transcription termination in gene constructs containing multiple tandem polyadenylation signals.

The processes of pre-mRNA 3'-end cleavage and polyadenylation have been closely linked to transcription termination by RNA polymerase II. We have studied the relationship between polyadenylation and transcription termination in gene constructs containing tandem poly(A) signals, at least one of which is the inefficient polyomavirus late poly(A) site. When identical tandem viral signals were separated by fewer than 400 bp, they competed for polyadenylation. The upstream site was always chosen preferentially, but relative site choice was influenced by the distance between the signals. All of these constructs showed the same low level of transcription termination as wild type polyomavirus, which contains a single late poly(A) site. When tandem poly(A) signals were not identical, a stronger downstream signal could outcompete a weaker upstream signal for polyadenylation without altering the efficiency of transcription termination characteristic for use of the upstream signal. Thus, if a weak polyoma virus late poly(A) signal (associated with inefficient transcription termination) preceded a strong rabbit beta-globin signal (associated with efficient transcription termination), termination remained inefficient, but the distal signal was most often chosen for polyadenylation. These results are consistent with independent regulation of polyadenylation and transcription termination in this system and are discussed in light of current models for the dependence of transcription termination on a functional poly(A) site.

3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes.

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

3' RNA Processing Efficiency Plays a Primary Role in Generating Termination-Competent RNA Polymerase II Elongation Complexes

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

Mammalian Mitochondrial DNA Sequences Can Function as in Vivo Bacterial Transcription Terminators

We have used a prokaryotic terminator identification vector, pDR721, to isolate regions from rat mitochondrial DNA (mtDNA) that can act as transcription terminators in vivo. Three independent fragments having terminator capability have been mapped to three general regions of the mitochondrial genome. Two terminators, pRMT1 and pRMT3, are found within and around the D-loop and cytochrome b gene, respectively, while the third, pRMT5, is located at the 3′-end of the 16S ribosomal RNA gene. After subcloning into host cells which carried temperature sensitive mutations in the termination factor, rho protein, galactokinase assays at the permissive and non-permissive temperatures suggested that pRMT3 acted as a rho-independent termination element while the other two, pRMTI and pRMT5, were dependent on rho protein (or a rho-like protein) for efficient transcription termination.

Characterization of the mouse beta maj globin transcription termination region: a spacing sequence is required between the poly(A) signal sequence and multiple downstream termination elements.

For the majority of mRNA encoding eukaryotic transcription units, there is little or no knowledge of the elements responsible for transcription termination or how they may interact with RNA polymerase. In this report, we have used recombinant adenovirus reporter vectors to characterize the mouse beta maj globin sequence elements that cause transcription termination. Within the globin 3' termination region, we have identified at least three sequence elements which induce significant levels of transcription termination (> 50%). The smallest functionally active element (64% termination) is 69 bp in length. The natural arrangement of these elements results in a cumulative termination which is greater than 90%. Recognition of the termination elements by RNA polymerase II depends on the presence of a functional poly(A) signal sequence. We demonstrate that efficient transcription termination depends on appropriate spacing between the poly(A) signal sequence and the termination element.

Characterization of the Mouse βmaj Globin Transcription Termination Region: a Spacing Sequence is Required Between the Poly(A) Signal Sequence and Multiple Downstream Termination Elements

For the majority of mRNA encoding eukaryotic transcription units, there is little or no knowledge of the elements responsible for transcription termination or how they may interact with RNA polymerase. In this report, we have used recombinant adenovirus reporter vectors to characterize the mouse beta maj globin sequence elements that cause transcription termination. Within the globin 3' termination region, we have identified at least three sequence elements which induce significant levels of transcription termination (> 50%). The smallest functionally active element (64% termination) is 69 bp in length. The natural arrangement of these elements results in a cumulative termination which is greater than 90%. Recognition of the termination elements by RNA polymerase II depends on the presence of a functional poly(A) signal sequence. We demonstrate that efficient transcription termination depends on appropriate spacing between the poly(A) signal sequence and the termination element.

Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.

Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycinresistance reporter gene

Several multicopy promoter-probe plasmid vectors have been constructed that replicate in Brevibacterium lactofermentum and related coryneform amino acid-producing bacteria. Transcriptional activity is detected by the expression of a promoterless aminoglycoside phosphotransferase gene ( kan) derived from transposon Tn5; expression of this gene confers kanamycin resistance in B. lactofermentum. An efficient transcriptional terminator from the B. lactofermentum trp operon has been inserted upstream of the kan coding region to prevent significant transcriptional readthrough from vector promoters. The cat gene from Streptomyces acrimycini or the hygromycin-resistance gene from S. hygroscopicus are used as primary selection markers in the promoter-probe plasmid vectors. Using the promoter-probe vectors described in this paper, we have cloned several transcriptionally active fragments from the endogenous plasmid pBL1 of B. lactofermentum into Escherichia coli and/or B. lactofermentum.

The involvement of transcriptional read-through from internal promoters in the expression of a novel endoglucanase geneFSendA, fromFibrobacter succinogenesAR1

Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1. Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1). The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1. Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression. Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination. The possibility of autogenous regulation by translation products is suggested.

Analysis of chloroplast promoters using bidirectional transcription vectors

Spinach chloroplast RNA polymerase has been shown to efficiently terminate transcription at the threonine attenuator (thra) from Escherichia coli. In this study, efficient transcription termination by the chloroplast RNA polymerase was observed at a second prokaryotic terminator, the histidine attenuator (hisa) from Salmonella typhimurium. Termination occurred regardless of the orientation of either attenuator. In higher-plant chloroplast DNA, the genes for the beta subunit of the ATPase (atpB) and the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) are adjacent and divergently transcribed. Bidirectional transcription vectors, containing the histidine and threonine terminators, were constructed to analyze the divergently oriented atpB and rbcL promotors. One plasmid construction, pRTT7, contained two tandem copies of the threonine attenuator (pRTT7). Two additional constructs, pRHT1 and pRHT2, each contained oppositely oriented copies of thra and hisa. A DNA fragment containing the rbcL and atpB promoters was inserted between the two terminators present in the pRTT7, pRHT1, and pRHT2 plasmids. Transcription of these recombinant DNAs by spinach chloroplast RNA polymerase resulted in discretely sized rbcL and atpB transcripts. In addition, these bidirectional transcription vectors were used to identify previously uncharacterized chloroplast promoters.

Stem-loop structures at the 3′ end of tobacco Rubisco large subunit mRNA

The are two inverted repeat nucleotide (nt) sequences, each capable of forming a stem-loop structure ( sls) at the 3′ end of the tobacco Rubisco large subunit mRNA ( rbcL). The smaller sls is followed by a larger sls. The in vivo functions of the 3′ sls of the rbcL mRNA were characterized using the Escherichia coli system. S1 mapping of the rbcL transcripts synthesized in E. coli revealed that the 3′ end of a major transcript in the bacterial cell is almost identical to the 3′ end of authentic chloroplast (cp) rbcL mRNA. This native 3′ end is located 4 nt downstream from the larger sls for the cp mRNA and 6 nt for the bacterial transcript, respectively. Deletion expriements show that the larger sls is essential for producing the native 3′ end of rbcL mRNA in E. coli. The sls do not function an an efficient transcription terminator but can stabilize upstream mRNA segments in vivo.

Chloroplast mRNA 3? end maturation is biochemically distinct from prokaryotic mRNA processing

We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro. In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction. The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract. We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination. The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts. We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in Gram-negative bacteria

To combine the features of the Ω interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposen, called Omegon-Km. The Omegon-Km transposen is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS 7. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the β interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene ( aphA) which is expressed well in many Gram-negative bacteria. The IS 7 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain m which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gramnegative soil and water bacteria ( Rhizobium leguminosarum, Paracoccus denitrifians) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

Structure and organization of the pel genes from Erwinia chrysanthemi EC16.

The pelA and pelC genes from Erwinia chrysanthemi EC16 were sequenced and overexpressed in Escherichia coli cells. These genes and two others from the same strain that were characterized previously encode catalytically related pectate lyase proteins that are involved with the maceration and soft-rotting of plant tissue. The pel genes of strain EC16 were organized as two loosely linked clusters, with two structurally homologous genes in each. The pelA/E cluster also contained the remains of an additional pel gene, the 5' portion of which had been removed by a prior deletion event. Each of the four functional pel genes but not the deleted one contained an efficient rho-independent transcriptional terminator after the translational stop. These and other data indicate that the pel genes are all independently regulated despite their structural homology and tandem clustered organization. Two of the genes, pelA and pelE, encoded proteins that differed greatly in their isoelectric points and ability to macerate plant tissue. A recombinant gene constructed with the 5' portion of pelE and the 3' portion of pelA yielded a chimeric protein with high pectate lyase activity but relatively low maceration activity. This result raised the possibility that the poor maceration ability of the pelA gene product may involve other properties in addition to its low isoelectric point.

The mouse ribosomal gene terminator consists of three functionally separable sequence elements.

The structural requirements for 3' end formation of mouse pre-rRNA have been studied. Three sequence elements are shown to be required for accurate and efficient transcription termination by RNA polymerase I (pol I) assayed both in a cell-free transcription system and in vivo after transfection of rDNA minigene constructs into 3T6 cells. The essential termination signal is the previously identified 18-bp conserved element (AGGTCGACCAGATTANTCCG) that contains a SalI restriction site. This sequence motif (the 'Sal box') interacts with a specific nuclear protein that directs transcription termination. Here we demonstrate that the 'Sal box' sequence motif is sufficient for termination of pol I transcripts and the release of the nascent RNA chains from the template. However, in addition to this termination signal, pyrimidine-rich sequences flanking the box at the 5' and 3' side play a role in the efficient and correct formation of authentic pre-rRNA termini. Downstream sequences contribute to the efficiency of the termination reaction, whereas the position of 3' end formation (i.e. 21 bp upstream of the 'Sal box') is affected by 5' flanking regions. These flanking regions are recognized by at least two different nuclear factors which specifically bind to DNA sequences located upstream and downstream of the 'Sal box'.

Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional terminators and useful for cloning of DNA from Streptococcus pneumoniae.

A new Escherichia coli plasmid cloning vector, pJDC9, was constructed by replacing the TcR determinant of pMB9 with the erythromycin-resistance ermB determinant and the lacZ alpha gene of pUC19. Efficient transcriptional terminator signals were positioned at both ends of lacZ alpha. Evidence is presented that protection of the vector by terminator signals enabled cloning of many fragments of DNA from Streptococcus pneumoniae that were unstable in vectors lacking such protection, including pBR322. At the pneumococcal mal locus, three promoter sites required such protection, while overexpression of the malX protein appeared to be lethal despite such protection.