mRNA Initiation Site Research Articles

Identification of a transcriptional silencer in the 5'-flanking region of the human epsilon-globin gene.

We have studied the 5'-flanking sequences required for the transcriptional regulation of human epsilon-globin gene expression. A series of deletion mutants of the human epsilon-globin gene 5'-flanking sequences were constructed and linked to the bacterial chloramphenicol acetyltransferase gene. Expression of these constructs was tested in HeLa cells and the human erythroleukemia K-562 cells. By measuring chloramphenicol acetyltransferase activities and mRNA levels we found that the sequence between -177 and -392 base pairs (bp) relative to the mRNA initiation site exerts a negative effect on epsilon-globin promoter activity. This effect is more pronounced in HeLa cells compared with K-562 cells. To further characterize the negative control region we cloned the DNA sequence between -177 and -392 bp either 5' or 3' of the epsilon-globin promoter and in either orientation. Our data indicate that this negative control region inhibits the epsilon-globin promoter activity in a position- and orientation-independent manner, thus suggesting that it is a silencer. In addition, the silencer also inhibits the expression from the Herpesvirus thymidine kinase promoter. Sequence comparison reveals that there are three short regions within the silencer that share extensive homology with those found in other negative control DNA elements. Our results therefore indicate that an upstream silencer element is present in the epsilon-globin gene and that it may play an important role in the control of epsilon-globin gene expression during development.

Evolution and transcription of Old World monkey globin genes

Although apparently inactive in the whole animal, the δ globin genes from three species of Old World monkey (rhesus, baboon and green monkey) are all functional in an in vitro transcription assay. Their activities in vitro are similar to that of the functional human delta gene. A fourth monkey gene, from the colobus monkey, is transcribed approximately fivefold less efficiently than the others. This reduced in vitro activity results from a 20 basepair deletion, which removes the normal site of mRNA initiation. When the deletion is repaired by site-directed mutagenesis, transcriptional activity increases to the level observed for the other δ genes. We also report the complete nucleotide sequences of the colobus β, colobus δ, and rhesus δ genes. Sequence comparisons show that the δ and β genes in the same species have not exchanged genetic information since the divergence of the human and monkey lineages. Phylogenetic analysis of these sequences affirms that the Old World monkey δ genes are evolving more rapidly than their functional counterparts. Moreover, the rate of replacement substitutions has risen to equal that of non-coding DNA, as expected for genes no longer under selective constraint.

The Pseudomonas oleovorans alkane hydroxylase gene: Sequence and expression

We have identified and sequenced the Pseudomonas OCT plasmid-encoded alkane hydroxylase gene (alkB) and its promoter. The transcription initiation site of the alkBAC mRNA was determined by nuclease SI mapping. A putative interaction site with RNA-polymerase was identified based on homology of the alk promoter with other Pseudomonas promoters. The alkB gene encodes a 401-amino acid polypeptide which, despite an unusual codon composition, can be expressed at high levels in Escherichia coli and Pseudomonas. The amino-terminal sequence of the purified cytoplasmic membrane alkane hydroxylase was determined and was found to be in agreement with the nucleotide sequence. The translation product of the alkB gene contains nine hydrophobic sequences of which eight are sufficiently long to be membrane-spanning segments. The amino-terminal sequence resembles that of several bacterial integral membrane proteins and is not cleaved off following translation.

Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and TN1O

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.

Efficient activation of transcription in yeast by the BPV1 E2 protein

The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level.

Analysis of a region of the human cytomegalovirus (AD169) genome coding for a 25-kilodalton virion protein

In a previous study, we reported the isolation of a lambda gt11 cDNA clone (C3) for a virus message that mapped to the HindIII R fragment (human cytomegalovirus [AD169]). In this report, we further analyze transcription from this region of the genome. C3 was used to probe Northern (RNA) blots of RNA isolated from infected cells. Two abundant messages, 1.3 and 1.6 kilobases (kb) in size, were detected at 62 h postinfection (p.i.). Examination of different time points determined that the 1.6-kb mRNA accumulated in infected cells between 24 and 48 h p.i. and was classified as a late message. The 1.3-kb message was transcribed early in infection and was initially detected around 12 h p.i. Both transcripts were suppressed when infected cells were treated with inhibitors of DNA synthesis. Sequencing and S1 analysis identified the 5' ends of these two messages within 240 nucleotides of each other. Two CAAT-TATA motifs were found upstream of the 1.3- and 1.6-kb mRNA initiation sites, which suggested that the promoters were also closely associated. Antisera made to the fusion protein, synthesized from the C3 clone, detected a 25-kilodalton (kDa) virus protein found in infected cells and purified virions. Western blot (immunoblot) analysis of infected-cell proteins at various times after infection demonstrated that the accumulation of the 25-kDa protein coincided with the appearance of the 1.6-kb message. Therefore, we conclude that the 25-kDa virion protein is translated from the 1.6-kb message.

Fine mapping of the immediate-early gene of the Indiana-Funkhauser strain of pseudorabies virus.

The immediate-early gene transcript of pseudorabies virus was found to be 5.6 kilobases and colinear with the DNA genome. Transcription initiation started in the BamHI E fragment, 45 nucleotides from the BamHI-E/BamHI-I junction. The first AUG codon was located in BamHI-I, 260 nucleotides from the mRNA initiation site, and a potential TGA termination codon was located in BamHI-J, 161 nucleotides from the BamHI-I/BamHI-J junction. The AATAAA polyadenylation signal was 30 bases from the stop codon, while the actual polyadenylation site was 18 bases further downstream.

Antiviral activity of conjugates between poly( l-lysine) and synthetic oligodeoxyribonucleotides

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell intemalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (β)-anomeric oligos to poly( l-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 μM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences i.e., to untranscribed regions. No antiviral activity can be obtained with (α)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.

A Discrete Element 3′ of Human Immunodeficiency Virus 1 (HIV-1) and HIV-2 mRNA Initiation Sites Mediates Transcriptional Activation by an HIV trans Activator

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Identification of two transcription factors that bind to specific elements in the promoter of the adenovirus early-region 4.

Two kinds of trans-acting factors that regulate transcription from the promoter of the adenovirus early-region 4 (E4) have been identified by reconstituting nuclear extracts of HeLa cells. They were designated E4TF1 and E4TF3 for E4 transcription factors. These factors were responsible for efficient and accurate transcription in vitro from the E4 promoter, as were another transcription factor, designated E4TF2, and a crude fraction containing endogenous RNA polymerase II. E4TF1 stimulated transcription from the E4 promoter but not from the major late promoter or the E4 mutant promoter lacking the E4TF1-binding site. Footprint analysis of E4TF1 revealed that it binds to a specific region, residing between 132 and 152 base pairs upstream from the initiation site of the E4 mRNA. E4TF3 also regulated transcription from the E4 promoter. E4TF3 protected four ca. 20-base-pair regions in a DNase I footprinting assay. They were located around 40, 160, 230, and 260 base pairs upstream from the initiation site of E4 mRNA. Specific inhibition of E4 transcription was observed by addition of DNA fragments covering one of the E4TF1- and E4TF3-binding sites to in vitro transcription assays. These results suggest that both E4TF1 and E4TF3 regulate E4 transcription by binding to the specific upstream elements in the E4 promoter. These factors may be involved in the E1A transactivation of E4 transcription.

Conservation in the 5′ flanking sequences of transcribed members of the Caenorhabditis elegans major sperm protein gene family

The major sperm proteins (MSPs) are encoded in the Caenorhabditis genome by a multigene family with more than 50 genes dispersed in small clusters at three chromosomal loci. In spite of their dispersed locations, all of the MSP genes appear to be expressed at the same time exclusively in the testis, indicating co-ordinate temporal and spatial regulation of these dispersed genes. Many of the MSP genes must be transcribed, because RNA hybridization with gene-specific probes showed that individual genes each contribute less than 3% to the total poly(A) + RNA, and 13 out of 14 sequenced cDNAs came from different genes. Primer extension assays from MSP mRNA showed that most of the MSP mRNAs must be initiated at position −35 from the translation start codon. Extensive similarity was found in the first 100 nucleotides of genomic sequence flanking the start codons of ten MSP genes from different chromosomal locations. All MSP genes contained a consensus ribosome binding site, a consensus TATA homology 27 nucleotides distal to the site of mRNA initiation, and ten highly conserved nucleotides adjacent to the site of initiation. All the MSP genes contained the sequence AGATCT located approximately 65 nucleotides upstream from the transcriptional start, but little or no similarity was found more distal to this. Some of these conserved sequences may be cis-acting control elements that ensure the cell and temporal specificity of transcription of these co-ordinately regulated genes.

An archaebacterial RNA polymerase binding site and transcription initiation of the hisA gene in Methanococcus vannielii.

Transcription initiation of the hisA gene in vivo in the archaebacterium Methanococcus vannielii, as determined by nuclease S1 and primer extension analyses, occurs 73 base pairs (bp) upstream of the translation initiation site. Binding of M. vannielii RNA polymerase protects 43 bp of DNA, from 35 bp upstream (-35) to 8 bp downstream (+8) of the hisA mRNA initiation site, from digestion by DNase I and exonuclease III. An A + T rich region, with a sequence which conforms to the consensus sequence for promoters of stable RNA-encoding genes in methanogens, is found at the same location (-25) upstream of the polypeptide-encoding hisA gene. It appears therefore that a TATA-like sequence is also an element of promoters which direct transcription of polypeptide-encoding genes in this archaebacterium.

The yeast PHO5 promoter: phosphate-control elements and sequences mediating mRNA start-site selection.

Transcription of PHO5 is strongly regulated in response to the level of inorganic phosphate (Pi) present in the growth medium. We have identified elements required for PHO5 expression by analyzing small deletions in the PHO5 promoter on chromosome II. The results reveal three functionally different components of the PHO5 promoter: regulatory regions, a "TATA" element, and specific mRNA initiation sites. The regulatory regions contain related 19-base-pair (bp) dyad sequences acting as phosphate-controlled upstream activation sites (UASpS). These UASpS mediate the transcriptional activation of PHO5 observed in low Pi conditions. The unlinked but coordinately regulated PHO11 promoter contains a single copy of an almost identical dyad sequence, suggesting that there is a common regulatory UASp for both genes. A TATA element is absolutely required for detectable PHO5 transcription. Specific purine-pyrimidine motifs (RRYRR) (R = purine and Y = pyrimidine) serve as PHO5 mRNA initiation sites, but only if they lie 55-110 bp downstream of a functional TATA element. Such an "initiation window" is not found in higher eukaryotes and implies mechanistic differences in the transcription machineries between yeast and higher eukaryotes.

Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site.

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression, but their efficient introduction within intact cells proved to be difficult to realize. As a step toward this goal, small (13- or 15-mer) synthetic oligodeoxyribonucleotides have been coupled at their 3' ends to epsilon-amino groups of lysine residues of poly(L-lysine) (Mr, 14,000). A 15-mer oligonucleotide-poly(L-lysine) conjugate complementary to the initiation region of vesicular stomatitis virus (VSV) N-protein mRNA specifically inhibits the synthesis of VSV proteins and exerts an antiviral activity against VSV when added in the cell culture medium at doses as low as 100 nM. Neither synthesis of cellular proteins nor multiplication of encephalomyocarditis virus was affected significantly by this oligonucleotide conjugate. The data suggest that oligonucleotide-poly(L-lysine) conjugates might become effective for studies on gene expression regulation and for antiviral chemotherapy.

Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription.

In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA. Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription. These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself. From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription.

In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA. Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription. These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself. From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.

The nucleotide sequence of the polyhedrin gene region from the multicapsid baculovirus of orgyia pseudotsugata

The nucleotide sequence was determined of a 1.6-kb restriction fragment containing the polyhedrin gene of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). In addition, both the 5' and 3' termini of the polyhedrin mRNA were located in the flanking sequences using S1 mapping procedures. 5′ Leader, 3′ flanking, and open reading frame sequences of 50–52, 145–167, and 735 nucleotides, respectively, were determined, for a total mRNA length of about 935 nucleotides. A potential 5′ regulatory sequence which included the mRNA initiation site was identified and an open reading frame upstream of the polyhedrin gene was located. In addition to the polyhedrin mRNA, two major transcripts upstream of the polyhedrin gene were identified by transcriptional mapping.