The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

A conditional block to transcriptional elongation is an important mechanism for regulating c-myc gene expression. This elongation block within the first c-myc exon was defined originally in mammalian cells by nuclear run-on transcription analyses. Subsequent oocyte injection and in vitro transcription analyses suggested that sequences near the end of the first c-myc exon are sites of attenuation and/or premature termination. We report here that the mapping of single stranded DNA in vivo with potassium permanganate (KMnO4) and nuclear run-on transcription assays reveal that polymerase is paused near position +30 relative to the major c-myc transcription initiation site. Deletion of 350 bp, including the sites of 3'-end formation and intrinsic termination defined in oocyte injection and in vitro transcription assays does not affect-the pausing of polymerase in the promoter-proximal region. In addition, sequences upstream of +47 are sufficient to confer the promoter-proximal pausing of polymerases and to generate the polarity of transcription farther downstream. Thus, the promoter-proximal pausing of RNA polymerase II complexes accounts for the block to elongation within the c-myc gene in mammalian cells. We speculate that modification of polymerase complexes at the promoter-proximal pause site may determine whether polymerases can read through intrinsic sites of termination farther downstream.

[1]  J. Lis,et al.  DNA sequence requirements for generating paused polymerase at the start of hsp70. , 1992, Genes & development.

[2]  M. Groudine,et al.  Transcription elongation and eukaryotic gene regulation. , 1990, Oncogene.

[3]  M. Mathews,et al.  HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation , 1989, Cell.

[4]  U. Siebenlist,et al.  Two distinct mechanisms of transcriptional control operate on c-myc during differentiation of HL60 cells , 1988, Molecular and cellular biology.

[5]  D. Bentley,et al.  A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription. , 1992, Genes & development.

[6]  S. Chen‐Kiang,et al.  Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Luciw,et al.  Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat. , 1989, Genes & development.

[8]  B. Wold,et al.  In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. , 1990, Science.

[9]  K. Jones.  HIV trans-activation and transcription control mechanisms. , 1989, The New biologist.

[10]  B. Cullen.  The HIV-1 Tat protein: An RNA sequence-specific processivity factor? , 1990, Cell.

[11]  D. Luse,et al.  Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing. , 1991, Genes & development.

[12]  S. Sasse-Dwight,et al.  Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor σ 54 , 1990, Cell.

[13]  N. Hay,et al.  Attenuation in the control of SV40 gene expression , 1982, Cell.

[14]  P. Luciw,et al.  Anti-termination of transcription within the long terminal repeat of HIV-1 by tat gene product , 1987, Nature.

[15]  J. Gralla,et al.  In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site. , 1989, Genes & development.

[16]  T Platt,et al.  Transcription termination and the regulation of gene expression. , 1986, Annual review of biochemistry.

[17]  E. Geiduschek,et al.  S. cerevisiae TFIIIB is the transcription initiation factor proper of RNA polymerase III, while TFIIIA and TFIIIC are assembly factors , 1990, Cell.

[18]  E. Ben‐Asher,et al.  Transcription of minute virus of mice, an autonomous parvovirus, may be regulated by attenuation , 1984, Journal of virology.

[19]  J. Gralla,et al.  Probing the Escherichia coli glnALG upstream activation mechanism in vivo. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Gallo,et al.  onc gene amplification in promyelocytic leukaemia cell line HL-60 and primary leukaemic cells of the same patient , 1982, Nature.

[21]  J. Gralla,et al.  Polymerase II promoter activation: closed complex formation and ATP-driven start site opening. , 1992, Science.

[22]  K. Jones,et al.  In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions. , 1989, Genes & development.

[23]  D. Price,et al.  Control of formation of two distinct classes of RNA polymerase II elongation complexes , 1992, Molecular and cellular biology.

[24]  J. Lis,et al.  RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene , 1991, Molecular and cellular biology.

[25]  D. Burton,et al.  The interaction of core histones with DNA: equilibrium binding studies. , 1978, Nucleic acids research.

[26]  C. Asselin,et al.  Molecular requirements for transcriptional initiation of the murine c-myc gene. , 1989, Oncogene.

[27]  K. Moberg,et al.  Isolation of a novel cDNA encoding a zinc-finger protein that binds to two sites within the c-myc promoter. , 1992, Biochemistry.

[28]  J. Roberts,et al.  Structure of transcription elongation complexes in vivo. , 1992, Science.

[29]  D. Price,et al.  Stability of Drosophila RNA polymerase II elongation complexes in vitro , 1992, Molecular and cellular biology.

[30]  B. Luckow,et al.  CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements , 1987, Nucleic Acids Res..

[31]  S. Collins,et al.  The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. , 1987, Blood.

[32]  K. Yamamoto,et al.  Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells. , 1978, Biochemistry.

[33]  S. Henikoff.  Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates. , 1990, Nucleic acids research.

[34]  Jeffrey W. Roberts.  Phage lambda and the regulation of transcription termination , 1988, Cell.

[35]  D. Luse,et al.  RNA polymerase II elongation complexes paused after the synthesis of 15- or 35-base transcripts have different structures , 1991, Molecular and cellular biology.

[36]  M. Groudine,et al.  Sequence requirements for premature termination of transcription in the human c-myc gene , 1988, Cell.

[37]  B. Berkhout,et al.  Tat trans-activates the human immunodeficiency virus through a nascent RNA target , 1989, Cell.

[38]  T. Kerppola,et al.  Intrinsic sites of transcription termination and pausing in the c-myc gene , 1988, Molecular and cellular biology.

[39]  R. Roeder,et al.  HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors. , 1992, Genes & development.

[40]  Mark Groudine,et al.  A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells , 1986, Nature.

[41]  J. Gralla,et al.  DNA supercoiling promotes formation of a bent repression loop in lac DNA. , 1987, Journal of molecular biology.

[42]  M. Groudine,et al.  Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter , 1992, Molecular and cellular biology.

[43]  H. Stunnenberg,et al.  Promoter melting by a stage-specific vaccinia virus transcription factor is independent of the presence of RNA polymerase , 1991, Cell.

[44]  Activation of HIV transcription by Tat , 1992 .

[45]  W. S. Hayward,et al.  The block to transcription elongation is promoter dependent in normal and Burkitt's lymphoma c-myc alleles. , 1990, Genes & development.