A TATA-like sequence located downstream of the transcription initiation site is required for expression of an RNA polymerase II transcribed gene.

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

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Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.302 ◽

1986 ◽

Vol 6 (1) ◽

pp. 302-314 ◽

Cited By ~ 50

Author(s):

R D Andersen ◽

B W Birren ◽

S J Taplitz ◽

H R Herschman

Keyword(s):

Rna Polymerase Ii ◽

Structural Gene ◽

Transcription Initiation ◽

Metal Ion ◽

Consensus Sequence ◽

Initiation Site ◽

Transcription Initiation Site ◽

Regulatory Sequences ◽

Base Pairs ◽

Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

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Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.302-314.1986 ◽

1986 ◽

Vol 6 (1) ◽

pp. 302-314

Author(s):

R D Andersen ◽

B W Birren ◽

S J Taplitz ◽

H R Herschman

Keyword(s):

Rna Polymerase Ii ◽

Structural Gene ◽

Transcription Initiation ◽

Metal Ion ◽

Consensus Sequence ◽

Initiation Site ◽

Transcription Initiation Site ◽

Regulatory Sequences ◽

Base Pairs ◽

Nuclease Mapping

As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene.

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A conserved sequence element is present around the transcription initiation site for RNA polymerase A inSaccharomycetoideae

Nucleic Acids Research ◽

10.1093/nar/12.2.1137 ◽

1984 ◽

Vol 12 (2) ◽

pp. 1137-1148 ◽

Cited By ~ 16

Author(s):

Martin Ph. Verbeet ◽

Jacobus Klootwijk ◽

Harm van Heerikhuizen ◽

Ruud D. Fontijn ◽

Erno Vreugdenhil ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Initiation Site ◽

Transcription Initiation Site ◽

Sequence Element ◽

Conserved Sequence

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The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2644 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2644-2652 ◽

Cited By ~ 43

Author(s):

S D Brown ◽

J Huang ◽

L H Van der Ploeg

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Initiation Site ◽

Transcription Initiation Site ◽

Protein Coding ◽

Pol Ii ◽

Protein Coding Genes ◽

Pol I ◽

Pol Iii ◽

Repetitive Protein

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

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The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2644-2652.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2644-2652

Author(s):

S D Brown ◽

J Huang ◽

L H Van der Ploeg

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Initiation Site ◽

Transcription Initiation Site ◽

Protein Coding ◽

Pol Ii ◽

Protein Coding Genes ◽

Pol I ◽

Pol Iii ◽

Repetitive Protein

All eukaryotic protein-coding genes are believed to be transcribed by RNA polymerase (Pol) II. An exception may exist in the protozoan parasite Trypanosoma brucei, in which the genes encoding the variant surface glycoprotein (VSG) and procyclic acidic repetitive protein (PARP) are transcribed by an RNA polymerase that is resistant to the Pol II inhibitor alpha-amanitin. The PARP and VSG genes were proposed to be transcribed by Pol I (C. Shea, M. G.-S. Lee, and L. H. T. Van der Ploeg, Cell 50:603-612, 1987; G. Rudenko, M. G.-S. Lee, and L. H. T. Van der Ploeg, Nucleic Acids Res. 20:303-306, 1992), a suggestion that has been substantiated by the finding that trypanosomes can transcribe protein-coding genes by Pol I (G. Rudenko, H.-M. Chung, V. P. Pham, and L. H. T. Van der Ploeg, EMBO J. 10:3387-3397, 1991). We analyzed the sequence elements of the PARP promoter by linker scanning mutagenesis and compared the PARP promoter with Pol I, Pol II, and Pol III promoters. The PARP promoter appeared to be of limited complexity and contained at least two critical regions. The first was located adjacent to the transcription initiation site (nucleotides [nt] -69 to +12) and contained three discrete domains in which linker scanning mutants affected the transcriptional efficiency: at nt -69 to -56, -37 to -11, and -11 to +12. The second region was located between nt -140 and -131, and a third region may be located between nt -228 and -205. The nucleotide sequences of these elements, and their relative positioning with respect to the transcription initiation site did not resemble those of either Pol II or Pol III promoter elements, but rather reflected the organization of Pol I promoters in (i) similarity in the positioning of essential domains in the PARP promoter and Pol I promoter, (ii) strong sequence homology between the PARP core promoter element (nt -37 to -11) and identically positioned nucleotide sequences in the trypanosome rRNA and VSG gene promoters, and (iii) moderate effects on promoter activity of mutations around the transcription initiation site.

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Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site.

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.653 ◽

1990 ◽

Vol 10 (2) ◽

pp. 653-661 ◽

Cited By ~ 116

Author(s):

A L Means ◽

P J Farnham

Keyword(s):

Rna Polymerase ◽

Binding Site ◽

Rna Polymerase Ii ◽

Dihydrofolate Reductase ◽

Transcription Initiation ◽

Simian Virus 40 ◽

Initiation Site ◽

Simian Virus ◽

Sequence Element ◽

Initiator Element

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

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Mapping of RNA polymerase on mammalian genes in cells and nuclei.

Molecular Biology of the Cell ◽

10.1091/mbc.3.10.1085 ◽

1992 ◽

Vol 3 (10) ◽

pp. 1085-1094 ◽

Cited By ~ 13

Author(s):

J Mirkovitch ◽

J E Darnell

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Initiation Site ◽

Isolated Nuclei ◽

Whole Cells ◽

Dna Template ◽

First Time ◽

Alpha Amanitin

The assembly of an RNA polymerase II initiation complex at a promoter is associated with the melting of the DNA template to allow the polymerase to read the DNA sequence and synthesize the corresponding RNA. Using the specific single-stranded modifying reagent KMnO4 and a new genomic sequencing technique, we have explored the melted regions of specific genes in genomic DNA of whole cells or of isolated nuclei. We have demonstrated for the first time in vivo the melting in the promoter proximal transcribed region that is associated with the presence of RNA polymerase II complexes. An interferon-inducible gene, ISG-54, exhibited KMnO4 sensitivity over approximately 300 nucleotides downstream of the RNA initiation site in interferon-treated cells when the gene was actively transcribed but not in untreated cells where the gene was not transcribed. The extent of KMnO4 modification was proportional to transcription levels. The KMnO4 sensitivity was retained when nuclei were isolated from induced cells but was lost if the engaged polymerases were further allowed to elongate the nascent RNA chains ("run-on"). The sensitivity to KMnO4 in isolated nuclei was retained if the run-on incubation was performed in the presence of alpha-amanitin, which blocks progress of engaged polymerases. A similar analysis identified an open sequence of only approximately 30 bases just downstream of the start site of the transthyretin (TTR) gene in nuclei isolated from mouse liver, a tissue where TTR is actively transcribed. This abrupt boundary of KMnO4 sensitivity, which was removed completely by allowing engaged polymerases to elongate RNA chains, suggests that most polymerases transcribing this gene paused at about position +20. The possibility of mapping at the nucleotide level the position of actively transcribing RNA polymerases in whole cells or isolated nuclei opens new prospects in the study of transcription initiation and elongation.

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A spectrum of mechanisms for the assembly of the RNA polymerase II transcription preinitiation complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.1049 ◽

1995 ◽

Vol 15 (2) ◽

pp. 1049-1059 ◽

Cited By ~ 10

Author(s):

C P George ◽

L M Lira-DeVito ◽

S L Wampler ◽

J T Kadonaga

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Selective Inhibition ◽

Initiation Site ◽

Tata Box ◽

Preinitiation Complex ◽

Basal Transcription ◽

Tata Box Binding Protein ◽

Basal Transcription Factors

To explore the diversity in the mechanisms of basal transcription by RNA polymerase II, we have employed a novel biochemical approach that involves perturbation of the transcription reaction with exogenously added TFIIB or TATA box-binding protein (TBP). Under these conditions, we observe promoter-selective inhibition of transcription by excess TFIIB or excess TBP. This inhibition occurs at the level of basal transcription, because it is observed with minimal promoters that comprise only the TATA box and initiation site sequences as well as with preparations of basal transcription factors that have been purified to greater than 90% homogeneity. In addition, the excess basal factors inhibit the assembly of a functional preinitiation complex but do not inhibit transcription initiation from preassembled preinitiation complexes. A study of several promoters revealed a reciprocal trend in the promoter specificity of inhibition by excess TFIIB versus that by excess TBP. At opposite ends of this spectrum, promoters are strongly inhibited by excess TFIIB but not excess TBP and vice versa. These results reveal the existence of a spectrum of mechanisms for preinitiation complex assembly at different promoters. The mechanistic preference appears to be specified by the aggregate of basal promoter elements rather than by an individual component, such as the TATA box or initiation site sequence. This spectrum provides a new parameter by which differences in the function of minimal class II promoters can be analyzed in the context of both basal and regulated transcription.

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P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.01859-07 ◽

2007 ◽

Vol 28 (3) ◽

pp. 1161-1170 ◽

Cited By ~ 83

Author(s):

Zhuoyu Ni ◽

Abbie Saunders ◽

Nicholas J. Fuda ◽

Jie Yao ◽

Jose-Ramon Suarez ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Processing ◽

Transcription Initiation ◽

Initiation Site ◽

The Body ◽

Elongation Complex ◽

Pol Ii ◽

Productive Elongation

ABSTRACT Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.

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