Efficient Transcription Initiation Research Articles

Construction of novel transcription vectors for production of biologically active transcripts from viral cDNAs.

A novel transcription vector, pUT118 which starts transcription to be directed to the natural 5' end of viral cDNA insert was constructed. After cleavage at a Xba I site just downstream of the cDNA insert, in vitro transcription can be performed by T7 RNA polymerase to produce full-length copies of viral RNAs. Biologically active CMV satellite RNA (Y strain) was successfully synthesized by inserting complete cDNA copy into a unique Nsi I site in pUT118 and transcribing the clone. To overcome the low transcription efficiency due to the modified T7 promoter in pUT118, pUT118GG was also created from pUT118 to add two extra G residues to the 5' end of the transcripts, allowing high efficiency of transcription initiation of the cDNA insert.

Significance of DNase I-hypersensitive sites in the long terminal repeats of a Moloney murine leukemia virus vector.

A Moloney murine leukemia virus-derived retroviral vector (N4) carrying the bacterial neomycin resistance gene (neo) was used to study the chromatin configuration of integrated proviral DNA in NIH 3T3-derived cell lines containing one copy of the vector DNA per cell. Three independently obtained cell lines were examined. In two of these cell lines, the vector was introduced by viral infection, while in the third the construct was introduced by DNA transfection. Such transfected cell lines (including the one examined) usually express 10- to 50-fold less virus-specific RNA than do cell lines obtained by viral infection. All three cell lines exhibited similar patterns of DNase I-hypersensitive (HS) sites. Two strong DNase I HS sites were detected in the 5' long terminal repeat, which contains signals required for proper and efficient initiation of viral transcription. One of these sites was found to overlap the viral enhancer sequences, while the other site mapped very close to the start site for viral transcription. A third HS site was detected in nearby internal viral sequences. Only one HS site was found in the 3' long terminal repeat, which contains the signal(s) required for proper addition of a poly(A) tail to viral transcripts. This HS site was located in the region of the viral enhancer. Several weak DNase I HS sites were also found in the cellular sequences adjacent to the integration sites, at different locations in each cell line analyzed. No common pattern of cellular DNase I HS sites was found. These observations suggest that the 5' and 3' long terminal repeats of integrated retroviral proviruses exhibit different chromatin conformations, possibly reflecting the different functions encoded by the otherwise identical sequences, and the DNase I HS sites detected in these studies reflect only a potential for transcription and are not a reflection of the high transcriptional activity characteristic of retroviruses.

Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter.

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.

Sequestration analysis for RNA polymerase I transcription factors with various deletion and point mutations reveals different functional regions of the mouse rRNA gene promoter.

We compared the ability of various deletion and substitution mutants of the mouse rRNA gene promoter to bind essential factors required for accurate transcription initiation by RNA polymerase I. Different amounts of a competitor template were first incubated with a mouse cell extract containing the whole complement of factors and RNA polymerase I, and then a tester template was added for the second incubation. Transcription was started by adding nucleoside triphosphates (one labeled), and the accurate transcripts were determined on a gel. The results indicated that the ability of 5' deletion mutants to sequester essential factors decreased almost concurrently with the impairment of in vitro transcription activity, whereas when the promoter sequence was removed from the 3' side, the transcription activity decreased earlier and more drastically than the sequestration ability. Similar, though not identical, results were obtained by preincubation with fraction D separated on a phosphocellulose column, indicating that the major factor which was sequestered was TFID, the species-dependent transcription initiation factor that binds first to the promoter in the initiation reaction (H. Kato, M. Nagamine, R. Kominami, and M. Muramatsu, Mol. Cell. Biol. 6:3418-3427, 1986). Compilation of the data suggests that a region inside the 5' half of the core promoter (-40 to -1) is essential for the binding of TFID. The 3' half of the promoter (-1 to downstream) is not essential for the binding of TFID but is highly important for an efficient transcription initiation. A strong down-mutant with a one-base substitution at -16 (G to A) had a reduced ability to bind to TFID, whereas a null mutant with a single base substitution at -7 (G to A) showed a binding ability similar to that of the wild-type promoter when tested with whole-cell extract. This null mutant, however, could not sequester the TFID well when incubated with fraction D alone, suggesting that the binding of TFID with this mutant is unstable in the absence of another factor(s) present in cell extract. The factor is not TFIA, which binds after TFID, because the addition of fraction A containing TFIA did not cause TFID to bind to the mutant. The availability of different mutants having lesions at different steps of transcription initiation will provide a powerful tool for the dissection of the initiation reaction of the RNA gene.

Fine structure genetic analysis of a beta-globin promoter.

A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis-acting sequences required for accurate and efficient initiation of beta-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter-containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

The core promoter of mouse rDNA consists of two functionally distinct domains.

We have determined the sequences constituting the minimal promoter of mouse rDNA. A very small region immediately upstream of the transcription start site (from -1 to -39) is sufficient to direct correct transcription initiation. Sequences immediately downstream of the transcription start site (+1 to +11) increase the efficiency of transcription initiation. Point mutations within the core promoter have been generated and assayed for their effects on template activity and on interaction with the pol I specific transcription factor TIF-IB. The core promoter element appears to consist of two functionally different domains. The distal sequence motif from position -22 to -16 is recognized by factor TIF-IB. Mutations within this region lead to similar changes of both template activity and binding of TIF-IB. Two point mutations within the proximal sequence motif from -15 to -1 do not affect TIF-IB binding although they severely impair transcription initiation. It is suggested, that this proximal region plays a role in the assembly of functional transcription initiation complexes rather than in the primary binding of TIF-IB.

The intragenic control region of the Xenopus 5 S RNA gene contains two factor A binding domains that must be aligned properly for efficient transcription initiation.

The in vitro recombination of deletion mutants of the Xenopus borealis somatic 5 S RNA gene allows construction of mutants containing substitutions of synthetic linker DNA for blocks of 9 or 10 nucleotides within the intragenic control region that binds transcription factor A. Two of the mutants containing exact linker replacements without insertion or deletion of nucleotides together alter 12 of 21 base pairs within the center of the control region. Both of these mutants bind factor A in an essentially normal pattern and support transcription, suggesting that a remarkable sequence variability can be tolerated in the center of the control region. The data obtained with these linker substitution mutants, along with data obtained previously with unidirectional deletion mutants, indicate that two short sequences at the boundaries of the control region serve as important recognition sites for the transcription factor. Additional closely related mutants containing slight deletions or insertions of form 2 to 10 base pairs do not bind factor A normally and do not support transcription in vitro. Thus, precise relative spacing of the boundaries of the control region is of critical importance for transcription.

Heterogeneity at the 5' termini of mouse dihydrofolate reductase mRNAs. Evidence for multiple promoter regions.

We have determined the sequence of the 1000 base pairs of DNA preceding the coding region of the mouse dihydrofolate reductase gene. 700 base pairs upstream of the translation start codon (position + 1) is the sequence CAACT, separated by 50 base pairs from the sequence TAATAA; these sequences resemble controlling elements responsible for accurate and efficient transcription initiation in a variety of eukaryotic genes. The region between -244 and -101 consists of a 3-fold tandem repeat of a 48-base pair sequence. S1 mapping results indicate that the 5' termini of the multiple dihydrofolate reductase mRNAs are heterogeneous, with the major terminus at -115 and with minor termini in the regions of -275 and -450. In addition, a portion of the 5'-ward region of the gene from -543 to -405 is represented differentially in some, but not all, mouse dihydrofolate reductase mRNAs. The above findings are consistent with the presence of two transcription promoter elements in the 5' end of the mouse dihydrofolate reductase gene. This has been substantiated by inserting portions of the 5'-ward 1000 base pairs of the gene into modular dihydrofolate reductase plasmids. When such constructs are transfected into Chinese hamster ovary cells lacking dihydrofolate reductase, function can be restored with equal transfection frequency when sequences surrounding the 5' CAACT-TAATAA region at -700 or a complete 48-base pair repeat from the region of -148 to -101 are present in the plasmid construct.

Efficiency of in vitro transcription of Dictyostelium discoideum actin gene is affected by the nucleotide sequence of the transcription initiation region.

The actin gene of Dictyostelium discoideum is transcribed faithfully but with very low efficiency in a cell-free system containing HeLa cell extract [Takiya, S., Tabata, T., Iwabuchi, M., Hirose, S., & Suzuki, Y. (1984) J. Biochem. (Tokyo) 95, 1367-1377]. Using the same in vitro system, we determined that the promoter activity of the actin 5 gene is 100-200 times weaker than that of the silkworm fibroin gene. To clarify the cause of the low transcription efficiency, various chimeric genes were constructed from the actin and fibroin genes, and their transcription efficiencies were examined in vitro. Both the TATA box and the transcription initiation site of the two natural genes functioned in the transcription of the chimeric genes, the efficiency of which was especially affected by the transcription initiation region. In chimeric genes having the upstream sequence of the actin gene and a downstream sequence including the transcription initiation site of the fibroin gene, the transcription efficiency was higher than one-third of that of the natural fibroin gene. In chimeric genes having the actin transcription initiation region and an upstream sequence of the fibroin gene, the transcription efficiency was as low as that of the natural actin gene. We concluded that the transcription initiation site is a part of the promoter and an essential region for directing faithful and efficient initiation of gene transcription.

Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates.

Two procedures were devised to examine initial events in the glucocorticoid-regulated transcription of integrated murine mammary tumor virus (MTV) DNA sequences. First, cells in monolayer culture were exposed for brief periods to dexamethasone and then permeabilized in situ with digitonin to allow entry of 32P-labeled ribonucleoside triphosphates and measurement of transcription rates. The results revealed that the rate of MTV RNA synthesis is stimulated selectively by dexamethasone after an apparent lag of approximately equal to 1 min and is half-maximal by 8-9 min. Second, the time course of transcription elongation was monitored by quantitating in single strand nuclease protection assays the accumulation of RNA sequences from different regions of the 7.8-kilobase MTV transcription unit. These experiments suggested that the rate of MTV RNA elongation in the presence of hormone does not differ substantially from elongation rates calculated for other genes transcribed by RNA polymerase II. Moreover, dexamethasone increased in parallel and to the same extent the levels of RNA from various segments of the transcription unit, implying that transcripts are elongated across the MTV element at a constant rate. We conclude that glucocorticoids stimulate MTV transcription solely via a rapid and selective increase in the efficiency of transcription initiation.

In monkey COS cells only the TATA box and the cap site region are required for faithful and efficient initiation of the fibroin gene transcription.

The DNA sequences necessary for faithful and efficient initiation of transcription of Bombyx mori fibroin gene have been studied in vivo using monkey COS cells and a SV40 origin vector. Transcriptional analysis of 5' deletion genes and of exact substitution genes including a series of single base substitution mutants indicates that the TATA box and the cap region are required for faithful and efficient initiation of the fibroin gene transcription in vivo, whereas far upstream sequences and the CAAT-like sequences are not required. In addition, single base substitution genes of the cap site region have clearly shown that there exists a strict sequence requirement of the cap region of the fibroin gene. These results define the basic promoter of the fibroin gene to the region spanning the TATA box and the cap site region.

Generation and functional analyses for base-substitution mutants of the adenovirus 2 major late promoter.

The function of guanosine residues surrounding the TATA box of the adenovirus 2 major late promoter (MLP) in promoting efficient transcription initiation and in selecting a specific transcription start site in vitro has been examined. Multiple and single base substitutions (G----A) were generated in this region (from -63 to +25 relative to the cap site, +1) of the MLP. The promoter activities of the wild type and 21 mutants were assayed in an in vitro transcription system using whole cell extract (WCE) prepared from HeLa cells. The results suggest that the strings of G residues immediately adjacent to the TATA box are not required for full promoter activity in vitro. These G residues also appear not to be involved in the selection of a specific transcription start site by RNA polymerase II in vitro, since the identical cap site was used by wild-type and mutated MLP's. However, two G residues (-55 and -57) were identified as part of an upstream promoter element: a G----A transition at either -55 or -57 resulted in a 2.6 fold reduction in promoter activity. However, neither single nor double G----A transitions at -62 and -63 had an effect on promoter activity.

Sequence Requirement for Transcription in vitro of the Human Corticotropin/β‐Lipotropin Precursor Gene

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase.

The catabolite activator protein (CAP) of Escherichia coli, complexed with cAMP, is required for efficient initiation of transcription from the galactose P1 promoter (start site at +1) but not from the overlapping P2 promoter (start site at -5) [Musso, R. E., DiLauro, R., Adhya, S. & deCrombrugghe, B. (1977) Cell 12, 847-854]. We investigated the interactions between CAP/cAMP and the gal promoter region in the presence of RNA polymerase. DNase I protection experiments of gal promoter restriction fragments revealed that CAP/cAMP protects the DNA from digestion between positions -50 and -25 and that RNA polymerase protects it from -35 to +10; however, gal DNA in the presence of both CAP/cAMP and RNA polymerase is protected from DNase I digestion between positions -68 and +15. Results of exonuclease III protection experiments show that RNA polymerase alone protects the gal DNA from -30 to +15; when both CAP/cAMP and RNA polymerase are present in the reaction, protection is afforded from -65 to +20. We directly quantified the amount of cAMP and CAP bound to gal promoter DNA in the presence of RNA polymerase by selectively pelleting the ternary complexes (CAP/cAMP-RNA polymerase-gal promoter DNA) in a Beckman Airfuge. We found two CAP molecules specifically bound to the gal promoter, although only one cAMP molecule was found in the complex at low cAMP concentrations (but sufficient to support P1 transcription). Thus, both the DNA protection experiments and the centrifugation results indicate that RNA polymerase induces the binding of a second CAP molecule to the gal promoter in forming stable initiation complexes. It appears that the second CAP molecule is needed to stimulate initiation from the P1 promoter; this may be involved in regulating the relative rates at which transcription begins from the two gal start sites.

RNA polymerase II ternary transcription complexes generated in vitro.

Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation.

Nucleotide sequence requirements for specific initiation of transcription by RNA polymerase I.

The nucleotide sequence(s) specifying RNA polymerase I initiation has been investigated by studying the transcription of deleted and nondeleted mouse ribosomal RNA gene (rDNA) templates in vitro. The deletion of 5'-flanking sequences upstream from position -- 39 did not affect transcriptional activity, but removal of sequences between positions -- 39 and -- 34 resulted in a 90% decrease of rDNA transcription. The template activity was completely eliminated by the further deletion of nucleotides -- 33 to -- 13. It is concluded that sequences between -- 34 and -- 12, upstream from the transcribed region, represent an essential control region for the initiation of transcription in vitro. Therefore, this region may be functionally analogous to the T-A-T-A box of RNA polymerase II promoters. In addition to this control region, sequences located further upstream (between positions -- 45 and -- 169) may also exert some function in efficient transcription initiation as revealed by competition experiments between wildtype and mutant rDNA templates.

Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription.

The role of the eucaryotic T-A-T-A box (Hogness box) homology sequence located approximately 30 base pairs upstream from the RNA initiation site has been further examined by oligodeoxynucleotide-directed site-specific mutagenesis. A method was employed which allows insertion of nucleotide-specific mutations into virtually any double-stranded, recombinant plasmid DNA. A synthetic mixed oligonucleotide, bearing defined multiple nucleotide substitutions at a single site, was used both as a specific mutagen during primed DNA repair synthesis in vitro, as well as a highly sensitive hybridization probe for the identification of the mutated cloned DNA. Using this methodology, an A leads to G transition mutation was introduced into the second position of the ovalbumin gene T-[A]-T-A box, and the effect of modifying this highly conserved nucleotide on the expression of the mutant DNA was analyzed in a cell-free transcription system. Comparison of two allelic ovalbumin genes, slightly divergent upstream from the TATA box, resulted in identical in vitro transcription efficiencies. While correct initiation of ovalbumin RNA transcripts was not affected, the efficiency of gene expression using the mutant template, compared to either the corresponding wild-type sequence or the allelic gene, was markedly reduced. These results suggest that it is the T-A-T-A box sequence which plays a role in the efficient initiation of RNA transcription in vitro and further supports the implication that this region may serve a promoter-related function in eucaryotic transcription.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.