DNA melting and promoter clearance by eukaryotic RNA polymerase I 1 1Edited by R. Ebright

Kahl, Brenda F.; Li, Han; Paule, Marvin R.

doi:10.1006/jmbi.2000.3743

Cited by 23 publications

(33 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…MPE·Fe is an intercalator that has been used extensively to study the interaction of duplex DNA-binding proteins with their respective substrates (36)(37)(38). MPE·Fe has also been used as a base pair-specific probe for structured RNAs (35).…”

Section: Discussionmentioning

confidence: 99%

“…Cleavage by MPE·Fe exhibits little sequence selectivity, a characteristic that makes it well-suited for protection studies. MPE·Fe footprinting has previously been used to determine nucleotides of DNA involved in complex formation with proteins and small molecules (36)(37)(38)(39). We surmised that the Figure 4A shows the results of an MPE·Fe footprinting reaction with the activator RNA in the presence of increasing amounts of dephospho-and phosphoPKR (lanes 4-6 and 7-9, respectively).…”

Section: The Rna-binding Activity Of Pkr Is Stimulated By Dephosphorymentioning

confidence: 95%

See 1 more Smart Citation

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Jammi

Beal

2001

Nucleic Acids Research

View full text Add to dashboard Cite

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho-and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: The Rna-binding Activity Of Pkr Is Stimulated By Dephosphorymentioning

confidence: 95%

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Jammi

Beal

2001

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…If RNA polymerase II has the capacity to close the downstream bubble, therefore, RNA polymerase II uses different contacts for closure than T. thermophilus RNA polymerase. Some chemical reactivity studies support a model of variable downstream template opening for multisubunit RNA polymerases (20,21), and functional studies indicate that the downstream bubble of human RNA polymerase II can interact with NTP substrates (12).…”

mentioning

confidence: 87%

A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors

Xiong

Burton

2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

When nucleoside triphosphate (NTP) substrates and ␣-amanitin are added to a human RNA polymerase II elongation complex simultaneously, the reaction becomes stalled in the core of the bond synthesis mechanism. The mode of stalling is influenced by NTP substrates at the active site and at downstream sites and by transcription factor IIF (TFIIF) and TFIIS. NTP substrates templated at i؉2, i؉3, and i؉4 downstream DNA sites can reverse the previously stable binding of an NTP loaded at the i؉1 substrate site. Deoxy-(d)NTPs and NDPs (nucleoside diphosphates) do not substitute for NTPs at the i؉2 and i؉3 positions (considered together) or the i؉4, i؉5, and i؉6 positions (considered together). The mode of stalling is altered by changing the number of downstream template sites that are accurately occupied by NTPs and by changing NTP concentration. In the presence of the translocation blocker ␣-amanitin, a steady state condition is established in which RNA polymerase II stably loads an NTP substrate at i؉1 and forms a phosphodiester bond but cannot rapidly complete bond synthesis by releasing pyrophosphate. These observations support a role for incoming NTP substrates in stimulating translocation; results appear inconsistent with the secondary pore being the sole route of NTP entry for human RNA polymerase II, and results indicate mechanisms of dynamic error avoidance and error correction during rapid RNA synthesis.A goal of transient state kinetic analyses is to reveal the internal mechanism of an enzyme reaction by observing synchronized millisecond events on a millisecond time scale. In this work, the mushroom toxin ␣-amanitin is utilized as a transient state inhibitor, locking human RNA polymerase II in the core of the elongation mechanism. By altering the conditions of stalling and by employing two distinct reaction quenching methods, details of the core mechanism of human RNA polymerase II become apparent. Recent x-ray crystal structures of Thermus thermophilus RNA polymerase elongation complexes indicate a simple thermal ratchet mechanism for elongation (1, 2), with nucleoside triphosphate (NTP) 2 substrates loading through the secondary pore, a solvent accessible channel, to the active site (designated iϩ1). Very little space is available in these structures to load NTPs through the main enzyme channel, which holds the DNA duplex and RNA-DNA hybrid. The downstream transcription bubble is closed at the iϩ2 position by base pairing, so it is difficult to imagine how NTP substrates could interact at iϩ2 or iϩ3 template sites. T. thermophilus ␤ Arg 422 makes a specific contact to the iϩ1 DNA template phosphate, and appears to provide a specific mechanism for closing the downstream bubble at the iϩ2 position. This mechanism for closing the downstream bubble is not conserved in human or yeast RNA polymerase II, in which the corresponding residue to ␤ Arg 422 is Rpb2 Gly 493 (human) and Rpb2 Gly 506 (yeast). Furthermore, even if the downstream bubble were open in the T. thermophilus structure, little space is available to...

show abstract

“…A, the isolated complexes were analyzed on a 28% polyacrylamide gel. Lanes 1, 3,5,7,9,11,13,15, and 17 show the RNA products of the isolated complexes stalled in the indicated registers. The higher mobility of the 5-nt RNA products in lanes 5,7,9,11,13,15, and 17 is due to the last incorporated nucleotide in the nascent RNA being an A instead of a G. Minor products in lanes 15 and 17 could be detected after longer exposure.…”

Section: Stalled Archaeal Transcription Complexes Contain a Homogenoumentioning

confidence: 99%

“…Lanes 1, 3,5,7,9,11,13,15, and 17 show the RNA products of the isolated complexes stalled in the indicated registers. The higher mobility of the 5-nt RNA products in lanes 5,7,9,11,13,15, and 17 is due to the last incorporated nucleotide in the nascent RNA being an A instead of a G. Minor products in lanes 15 and 17 could be detected after longer exposure. When the isolated complexes were chased by the addition of all NTPs (40 M each), no RNA products could be detected in lanes 2,4,6,8,10,12,14,16, and 18 indicating that all isolated complexes remained in a transcriptionally competent state.…”

Section: Stalled Archaeal Transcription Complexes Contain a Homogenoumentioning

confidence: 99%

Analysis of the Open Region and of DNA-Protein Contacts of Archaeal RNA Polymerase Transcription Complexes during Transition from Initiation to Elongation

Spitalny

Thomm

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation. We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation. RNAP was stalled between registers ؉5 and ؉20 using C-minus cassettes. The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position ؉5. Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register ؉8. A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers ؉6/؉7. A major transition was observed at registers ؉10/؉11. In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance. Between registers ؉11 and ؉20 both RNAP and transcription bubble moved synchronously with RNA synthesis. The distance of the catalytic center to the front edge of the exo III footprint was ϳ12 nucleotides in all registers. The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides. For complexes stalled between positions ؉10 and ؉20 the size of the transcription bubble was around 17 nucleotides. This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II.Transcription initiation requires formation of a preinitiation complex (PIC), 1 melting of DNA, formation of the first phosphodiester bonds, and promoter clearance involving movement of the open DNA region ("transcription bubble") and RNA polymerase. Finally, a stable ternary elongation complex is formed. These steps have been extensively studied during the last 2 decades in bacterial RNA polymerase and eukaryotic polymerase II (for reviews see Refs. 1 and 2) and to less extent in eukaryotic RNA polymerase III (3, 4) and RNA polymerase I (5) systems. In Archaea, open complex formation at the Methanococcus tRNA Val (6) and at the 16 S rRNA promoter of Sulfolobus (7) have been studied. The transition from initiation to elongation has not yet been investigated in Archaea.In bacteria, promoter isomerization from closed to open complex catalyzed by the predominant RNA polymerase holoenzyme (␤␤Ј␣ 2 70 ) occurs spontaneously in a temperature-dependent manner (8, 9). By contrast, nuclear RNA polymerase II (pol II; see Ref. 10) and Escherichia coli RNA polymerase specific for promoters of genes involved in nitrogen metabolism (␤␤Ј␣ 2 54 ; see Ref. 11) require ATP hydrolysis for promoter melting. In the pol II system promoter opening involves the helicase activity of TFIIH (12). Eukaryotic nuclear RNA polymerases I and III share with the 70 containing E. coli RNA polymerase the ability to produce an open complex of ...

show abstract

DNA melting and promoter clearance by eukaryotic RNA polymerase I 1 1Edited by R. Ebright

Cited by 23 publications

References 61 publications

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

A Tunable Ratchet Driving Human RNA Polymerase II Translocation Adjusted by Accurately Templated Nucleoside Triphosphates Loaded at Downstream Sites and by Elongation Factors

Analysis of the Open Region and of DNA-Protein Contacts of Archaeal RNA Polymerase Transcription Complexes during Transition from Initiation to Elongation

Contact Info

Product

Resources

About