Insertional mutagenesis of a plasmid-borne Escherichia coli rpoB gene reveals alterations that inhibit beta-subunit assembly into RNA polymerase

Landick, Robert; Colwell, A; Stewart, J

doi:10.1128/jb.172.6.2844-2854.1990

Cited by 21 publications

(17 citation statements)

References 67 publications

(46 reference statements)

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“…The amplified rpoC fragment was treated with XhoI and SalI (SalI cuts at rpoC codon 877 and is compatible with XhoI) and ligated into the XhoI site of the rpoB expression plasmid pRL706 to yield pKS1000 (Table I). pRL706 contains a modified BamHI (filled in) to SacI fragment from pRL385 (9) between the NcoI (filled in) and SacI sites of the lacI P trc plasmid pTrc99c (10). The modification places a unique XhoI site encoding Leu-Glu directly after the last codon of rpoB and before a His 6 tag, so that insertion of the XhoI-SalI fragment in pKS1000 fused the C-terminal GAG(Glu) codon of rpoB to the Nterminal GTG codon of rpoC (encodes Val in fusion), separated only by the XhoI site encoding Leu-Glu.…”

Section: Methodsmentioning

confidence: 99%

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Severinov¹,

Mooney²,

Darst³

et al. 1997

Journal of Biological Chemistry

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The rpoB and rpoC genes of eubacteria and archaea, coding, respectively, for the ␤ and ␤-like subunits of DNA-dependent RNA polymerase, are organized in an operon with rpoB always preceding rpoC. Here, we show that in Escherichia coli the two genes can be fused and that the resulting 2751-amino acid ␤::␤ fusion polypeptide assembles into functional RNA polymerase in vivo and in vitro. The results establish that the C terminus of the ␤ subunit and the N terminus of the ␤ subunit are in close proximity to each other on the surface of the assembled RNA polymerase during all phases of the transcription cycle and also suggest that RNA polymerase assembly in vivo may occur co-translationally. DNA-dependent RNA polymerase (RNAP)1 is the central enzyme of gene expression and a major target for regulation. Cellular RNAPs are large, multisubunit protein complexes. Core RNAP of Escherichia coli (ϳ380 kDa) contains four polypeptides: ␤Ј (155 kDa), ␤ (150 kDa), and a dimer of ␣ (37 kDa). Core RNAPs from eukaryotes contain 12 or more subunits with a total mass in excess of 600 kDa (1). The subunit composition of archaeal, chloroplast, and some eubacterial RNAPs is even more complex due to splits in the genes coding for the largest subunits (2, 3). Despite these differences, all cellular RNAPs exhibit striking and co-linear sequence similarities with the bacterial subunits. The two largest subunits comprise 60% of the RNAP mass and appear principally responsible for most of the enzyme's functions.The synthesis of RNAP subunits is coordinately regulated (4), but the exact mechanisms are unknown. In eukaryotes, many genes coding for RNAP subunits have similar regulatory elements, suggesting coordinated expression (5). In eubacteria and archaea, genes coding for the ␤-and ␤Ј-like subunits are organized in an operon; the gene coding for the ␤-like subunit always precedes that coding for the ␤Ј-like subunit (6). In eubacteria, the two genes are separated by a short, untranslated linker; in archaea they overlap by several codons (7).In this work we demonstrate that the E. coli rpoB and rpoC genes, coding, respectively, for the 1342 amino acid-long ␤ and the 1407-amino acid-long ␤Ј subunits, can be fused and that the resulting ␤::␤Ј fusion protein assembles into functional RNAP in vivo and in vitro. Furthermore, when a hexahistidine tag (His 6 tag) is inserted at the fusion site between ␤ and ␤Ј, the resulting RNAP can be immobilized on a sorbent containing Ni 2ϩ ions and is transcriptionally active in the immobilized state. On the basis of these data we conclude that the C terminus of ␤ and the N terminus of ␤Ј are within a very short distance of each other on the surface of the RNAP molecule during all phases of transcription.

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Section: Methodsmentioning

confidence: 99%

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Severinov¹,

Mooney²,

Darst³

et al. 1997

Journal of Biological Chemistry

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“…Much work remains before we can unambiguously assign functions or particular contacts within the transcription complex to the different regions of f~' described here, and those described previously for ~ Landick et al 1990a;Sagitov et al 1993). Our substitution analysis of ~ and ~' offers targets about which to ask more detailed questions that ultimately should make conclusive assignments possible.…”

Section: Implications For the Mechanism Of Terminationmentioning

confidence: 99%

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

Weilbaecher¹,

Hebron²,

Feng³

et al. 1994

Genes Dev.

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118

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To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the 13' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of [3', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of 13'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of [3'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different p-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.[Key Words: rpoC gene; [3' subunit; RNA polymerase; transcriptional termination] Received July 26, 1994; revised version accepted October 13, 1994.Escherichia coli RNA polymerase contains a core of four subunits: 6', 155 kD; [3, 150 kD; and two c~, 43 kD each. These subunits form a scaffold and catalytic center for synthesis of RNA on a double-stranded DNA template that appear to be conserved from bacteria to humans. The two largest subunits, 6' and B in E. coli, display significant sequence similarity to homologous subunits found in all multisubunit RNA polymerases (Allison et al. 1985; Jokerst et al. 1989; Young 1991 and references therein): Each contains eight to nine conserved, colinear segments, although no sequence conservation is evident between f3' and B. However, we currently lack a clear picture of how these conserved primary sequence motifs are positioned in the three-dimensional structure of RNA polymerase.In the transcription elongation complex (for review, see Das 1993;Chamberlin 1994;Chan and Landick 1994), RNA polymerase contacts the DNA over a 25-to ~Present address:

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“…The samples were then electrophoresed through a 10-20% gradient polyacrylamide SDS gel (Novex) and compared with the CNBr fragments of purified ␤ subunit. ␤ subunit was purified by preparative SDS͞PAGE of inclusion bodies from a 500-ml saturated culture of JM109 pRL385 (35) …”

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confidence: 99%

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

Wang¹,

Severinov²,

Landick³

1997

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

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RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all -independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3 end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to ␤904-950 replaced crosslinking to ␤ and to other parts of ␤ that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3 end in the paused complex and be related to events at -independent terminators.The his leader pause hairpin is a well characterized nascent RNA structure that modulates RNA chain elongation (1-4). It forms concomitantly with a paused conformation of the transcription complex midway through the his biosynthetic operon leader region, where it increases the lifetime of the paused complex at least 6-fold. The pause allows time for a ribosome to initiate synthesis of the leader peptide and release the paused polymerase (probably by disrupting the pause hairpin), thereby synchronizing ribosome and polymerase movement during transcriptional attenuation (5). This and other pause sites are regulatory timing mechanisms that both prevent transcription beyond a region where regulatory input is effective and put polymerase in the proper conformation to interact with regulatory molecules.Similar hairpins are involved in some, but not all, pause signals; are required at -independent terminators (6-8), where they trigger dissociation of the transcription complex; and alone (e.g., HK022 put; ref. 9) or in association with proteins (e.g., N-nut complex; ref. 8) can function as antiterminators, which modify the transcription complex to block recognition of both pause sites and terminators. Nascent RNA hairpins also are components of some eukaryotic regulatory mechanisms (e.g., HIV TAR RNA; ref. 10) and can trigger pausing or termination by RNA polymerase II in vitro (11). Although RNA hairpin-RNA polymerase interactions have long been suggested to play roles in pausing (12), termination (13-15), and antitermination (16), no direct evidence for these interactions or their effects exists.The his pause RNA hairpin offers an excellent paradigm to study these interactions. It is one of four components of the multipartite his pause signal that also depends on a 3Ј-proximal region of RNA or DNA, alignment of the 3Ј-terminal nucleotide and the incoming NTP, and duplex DNA that contacts polymerase downstream from the active site (1, 2). Both the his pause signal and -independent terminators ...

show abstract

Insertional mutagenesis of a plasmid-borne Escherichia coli rpoB gene reveals alterations that inhibit beta-subunit assembly into RNA polymerase

Cited by 21 publications

References 67 publications

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Tethering of the Large Subunits of Escherichia coli RNA Polymerase

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

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