Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II)

Gunderson, Samuel; Chapman, Kenneth A.; Burgess, Richard R.

doi:10.1021/bi00380a007

Cited by 94 publications

(79 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…By contrast, there was no protection on the noncoding strand (top strand) in the presence of GTP or both GTP and ATP. These results are consistent with those reported for other T7 polymerase-promoter complexes (Basu and Maitra, 1986;Smeekens and Romano, 1986;Gunderson et al, 1987). The exact upstream boundary of the footprint on the bottom strand could not be determined due to the inability of DNase I to bind and incise near the termini of a helix.…”

Section: Xbal Haelh T Ecori Hinflsupporting

confidence: 90%

“…The exact upstream boundary of the footprint on the bottom strand could not be determined due to the inability of DNase I to bind and incise near the termini of a helix. We assumed that the polymerase protected up to -20 at the upstream end based on previously reported results (Basu and Mai tra, 1986;Ikeda and Richardson, 1986;Gunderson et al, 1987). or when the XL-138mer was used as the template (data not shown).…”

Section: Xbal Haelh T Ecori Hinflmentioning

confidence: 99%

“…The upstream boundary of the protected region on the coding strand was assumed to be -20 based on other reported results (Basu and Maitra, 1986;Ikeda and Richardson, 1986;Gunderson et al, 1987). The initia tion complex formation requires the presence of GTP, the initiation nucleotide, and is inhibited by high NaCl concentration.…”

Section: A) Initiation Complexmentioning

confidence: 99%

See 2 more Smart Citations

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

Shi¹

1988

View full text Add to dashboard Cite

This thesis czr.sists of three main parts and totally eight ohacters. Ir. part I, I will present studies en the photochemistry ::psoraler.-DIIA adducts, specifically, the wavelength dependencies f:r the phot:reversals cf thyraidine-riMT (4' -hydrcxymethyi-4, :', 3-trir.er.thyipsoraien) mcr.cadducts and diadduct and the sarr.e adoucts incorporated in DMA helloes and the wavelength decer.decies for the ph:-ccorcsslir.kir.g cf thymidine-HMT mcr.cadducts in dcuhie-stranded helloes.In part II, I will report seme biological effects of psoralen-:;:".-, aiduccs, i.e., the effects on double-stranded DMA stability, DNA struoture, and transcription by E. coli and T7 RNA polymerases. Finally, Iwill focus on the applications of psoralen-DMA photochemistry to inves tigation cf protein-DIiA interaction during transcription, which includes the interaction cf E. coli and T7 RNA polymerases with D:;A in elongation complexes arrested at specific pscraien-DHA acduct sites as revealed by DMase I foctprinting experiments. DEDICATIONThis work is dedicated to Theresa Ng.

show abstract

Section: Xbal Haelh T Ecori Hinflsupporting

confidence: 90%

Section: Xbal Haelh T Ecori Hinflmentioning

confidence: 99%

See 1 more Smart Citation

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

Shi¹

1988

View full text Add to dashboard Cite

show abstract

“…Footprinting studies with methidiumpropyl-EDTAFe(II) indicate that the T7 polymerase protects the region from Ϫ17 to Ϫ4 (8,9). Methylation and ethylation interference studies indicate that the major groove of the promoter between Ϫ5 and Ϫ12 is important for polymerase binding (10).…”

mentioning

confidence: 99%

Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos

Shin

Kim

Cantor

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A full set of SP6 promoter variants with all possible single substitutions at positions ؊17 to ؉5 was constructed. Transcription activities of these variants were individually measured in vivo and in vitro to determine the contribution of each base pair to the promoter activity. The in vivo activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues ؊11, ؊9, ؊8, ؊7, and ؉1 (initiation site). All substitutions at ؊11, ؊9, ؊8, and ؊7 abolished formation of closed complexes, except for A؊8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition in vitro. In contrast, the in vitro activities of some other variants, measured on linearized templates, were different from those in vivo. Some variants at ؊13, ؊4, and ؊2, among others, showed exceptionally higher activities in vivo than in vitro, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A؊3T variant showed much lower activity in vivo than in vitro, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity. The bacteriophage SP6 genome encodes a single-subunit RNA polymerase that is very similar to the T7, T3, and K11 RNA polymerases. SP6 promoters consist of a highly conserved 20-bp sequence that extends from positions Ϫ17 to ϩ3 and exhibits a strong homology to the T7, T3, and K11 promoter sequences (1). Despite their homology, each polymerase shows highly stringent specificity for its own promoter sequence. Mutational studies with phage T7 promoters have yielded detailed information about the relationship of structure to function and the recognition of them by RNA polymerase. The promoter consists of two domains: an initiation domain downstream of Ϫ4 and a binding domain upstream of Ϫ5 (2, 3). Single base changes in the initiation domain have little effect on promoter binding but reduce the rate of transcription initiation. In contrast, changes in the binding domain reduce the efficiency of promoter binding but have little effect on the initiation of transcription. Base pairs at positions Ϫ11 through Ϫ7 of the T7 promoter are essential to the binding (4-7).The interactions of phage RNA polymerases with their promoters have also been studied by a variety of biochemical methods. Footprinting studies with methidiumpropyl-EDTAFe(II) indicate that the T7 polymerase protects the region from Ϫ17 to Ϫ4 (8, 9). Methylation and ethylation interference studies indicate that the major groove of the promoter between Ϫ5 and Ϫ12 is important for polymerase binding (10). More recent studies with base analog substitutions reveal that, at positions Ϫ11 and Ϫ10 of the T3 and T7 promoters, their RNA polymeras...

show abstract

“…Determination of K d s. Gel shifts were quantified with a PhosphorImager, and the K d s of monomer and dimer binding to PIE RNA or the K d of monomer binding to U1 RNA was calculated as previously described (9,35 Polyadenylation assay and peptides. The polyadenylation assay was performed as described before (10,11).…”

Section: Methodsmentioning

confidence: 99%

Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

Gunnewiek

Hussein

Aarssen

et al. 2000

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoproteinspecific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3 untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented.The U1 small nuclear ribonucleoprotein (snRNP) particle is the most abundant member of the spliceosomal snRNPs. Human U1 snRNP is comprised of 10 proteins and the 164-nucleotide U1 small nuclear RNA (U1RNA) and is required for splicing of pre-mRNA (38). One of the U1 snRNP-specific proteins, the U1A protein, contains two evolutionarily conserved RNA recognition motifs (RRMs) characteristic of a large family of proteins involved in the biosynthesis of cellular RNA (reviewed in reference 37). The signature motifs for the RRM family consist of two ribonucleoprotein (RNP) sequences, RNP1 and RNP2, which are the most conserved features of this family. The N-terminal RRM of U1A is, together with some flanking amino acids, necessary and sufficient for binding to the loop part of stem-loop 2 (SL2) sequence AUUGCAC of U1RNA (22,27,28). The structure of the N-terminal RRM of the U1A protein (amino acids [aa] 2 to 95) has been solved both by X-ray crystallography and by nuclear magnetic resonance (NMR) and consists of a ␤ 1 ␣ 1 ␤ 2 ␤ 3 ␣ 2 ␤ 4 structure in which the ␤ strands form a sheet with the highly conserved RNP1 and RNP2 motifs located in the two central ␤ strands, ␤ 3 and ␤ 1 , respectively (14, 23). An additional ␣ helix (helix 3; hereafter referred to as helix C) is present when a longer fragment of the U1A protein is analyzed (aa 2 to ...

show abstract

Interactions of T7 RNA polymerase with T7 late promoters measured by footprinting with methidiumpropyl-EDTA-iron(II)

Cited by 94 publications

References 19 publications

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos

Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

Contact Info

Product

Resources

About