John P. Richardson

Rho-dependent termination and ATPases in transcript termination

Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA-binding domain (RNA-BD) of known structure as well as an ATP-binding domain (ATP-BD) for which a structure has been proposed based on homology modeling. A model is proposed in which Rho first makes an interaction with a nascent RNA on a C-rich, primarily single-stranded rut region of the transcript as that region emerges from the exit site of RNA polymerase. A subsequent step involves a temporary release of one subunit of the hexamer to allow the 3 segment of the nascent transcript to enter the central channel of the Rho ring. Actions of the Rho structure in the channel on the 3 segment that are coupled to ATP hydrolysis pull the RNA from its contacts with the template and RNA polymerase, thus causing termination of its synthesis.

ATP-induced changes in the binding of RNA synthesis termination protein rho to RNA

The interaction between rho protein, an RNA synthesis termination factor from Escherichia coli , and synthetic RNA polymers has been studied using a membrane filter binding technique and by the inhibition of enzymes that digest RNA. The binding studies show that rho forms a very stable, salt-resistant complex with poly(C). The high affinity of the interaction correlates with the high activity of poly(C) as an effector of rho-ATPase. Poor rho-ATPase effectors, such as poly(U) and poly(A) also bind to rho but with much lower affinities. However, good binding is not a sufficient condition for activation of rho-ATPase; poly(dC) binds to rho with a higher affinity than poly(U) yet it does not activate rho-ATPase. At low ratios of rho to poly(C), one poly(C) molecule is bound for each molecule of hexameric rho. At higher ratios, a single poly(C) can bind several rho molecules independently. The sites for rho on poly(C) become staturated at a level of one rho molecule per 100 nucleotides, which is about the level expected for close packing of rho on a single-stranded RNA. From the size of poly(C) protected by rho against ribonuclease A digestion, it is concluded that the binding site on rho is large enough to contain 60 residues of poly(C). Poly(C) bound to rho is subject to a bimolecular displacement reaction by free poly(C). The rate of that reaction is decreased by ATP and β-γ imido ATP and increased by ADP. The tight binding of rho to poly(C) or poly(U,C) inhibits the action of polynucleotide phosphorylase, bovine brain exonuclease and Q β replicase on the RNA. The addition of ATP increases the effectiveness of rho in inhibiting these enzymes by a factor of two. ATP also decreases the maximum amount of rho that can be bound to poly(C) by a factor of two. However, ATP does not change the binding stoichiometry when sites on poly(C) are not limiting nor the amount or size of poly(C) protected from ribonuclease A digestion. A model to explain these effects plus the effects of nucleotides on the poly(C) displacement reaction is proposed in which ATP causes poly(C) to become wrapped around rho. We suggest that this occurs through multiple interactions at secondary RNA binding sites on rho.

Preventing the synthesis of unused transcripts by rho factor

John P. Richardson Department of Chemistry Programs in Biochemistry and Molecular, Cellular and Developmental Biology Indiana University Bloomington, Indiana 47405 Transcriptional terminators have three important functions in gene expression. One is as asignal that defines the end of transcription unit for a gene or a group of genes. These operonic terminators are essential elements of gene orga- nization because they allow for the totally independent expression of adjacent sequences on a DNA molecule. In addition, terminators are found either between genes expressed under control of a single promoter or in the region between the promoter and the first gene of the operon. These intergenic terminators serve to modulate the relative level of expression of different genes in an operon or even the whole operon itself, as in the case of attenuator regulation. Finally, terminators have also been found within genes. However, these intragenic terminators are latent and func- tion only when translation becomes uncoupled from tran- scription either by a mutational change in the gene or by certain forms of metabolic stress, such as starvation for an amino acid. They thus prevent continued synthesis of an unused transcript. Although intragenic terminators have been known to exist for fifteen years, details of their properties have been largely ignored. Some recent papers on the analysis of details of the structure and function of several representative examples of intragenic terminators in E. coli has focused attention again on their role and on the mechanistic properties that give them their latency (Wek et al., 1987; Ruteshouser and Richardson, 1989; Tsurushita et al., 1989; Alifano et al., 1991). Transcripts synthesized by action of E. coli RNA poly- merase are terminated by two distinct mechanisms (re- viewed by Yager and von Hippel, 1987; Platt, 1988). One involves a spontaneous release of an RNA molecule at a particular set of sequences; the other requires the action of an RNA release factor called rho. Over most sequences, transcriptional elongation is highly processive-the na- scent RNA is very stably attached in its complex with RNA polymerase and the DNA template. However, at certain limited sets of sequences, this stability drops to the point that the RNA molecules are spontaneously released. These intrinsic terminators are characterized by 40 bp of DNA that start with a GC-rich segment with an interrupted dyad symmetry followed by about six adenosine residues on the template strand. Release occurs when the tran- script has been extended to the end of those residues. The details of why this particular set of sequences serve intrinsically to terminate transcription have been analyzed in depth (Yager and von Hippel, 1991) and are the focus of further intensive studies (Reynolds et al., 1991).

Stabilization of the hexameric form of Escherichia coli protein rho under ATP hydrolysis conditions.

Abstract The state of oligomerization of Escherichia coli transcription termination protein rho has been studied under various conditions by velocity sedimentation, gel filtration and protein cross-linking. The sizes of the cross-linked products formed after reaction of rho with dimethyl suberimidate or dithiobis(succinimidyl propionate) indicate that it can exist as a hexamer of its single polypeptide. However, the results of the sedimentation and gel filtration studies indicate that rho readily dissociates into free monomers (with M r = 50,000) and the fraction of molecules in the hexameric form depends on the conditions. Although rho appears to be always partially dissociated whenever it is free, it migrates distinctly as a hexamer when it is bound to poly(C) and is primarily, if not exclusively, in that form whenever it catalyzes the hydrolysis of ATP. These conditions of hydrolysis include: at very low rho concentration (~1 μg/ml), which favors complete dissociation of free rho; in 0.5 m -KCl, which destabilizes the binding to poly(C) and favors the dissociation of free rho; and with a mutationally altered rho ( ts15 ), which in the free form is more fully dissociated than wild-type rho. We conclude from these results that the functionally active form of rho is a hexamer which has a molecular weight of 288,000 ± 12,000.

STRUCTURAL ORGANIZATION OF TRANSCRIPTION TERMINATION FACTOR RHO

Escherichia coli has two known modes for termination of RNA transcription (1–4). One is intrinsic to the function of RNA polymerase, which can spontaneously terminate transcription in response to certain, limited sequences. The other mode is dependent upon the action of an essential protein factor called Rho and occurs at sequences that are specific for its function but that are less constrained than the sequences for intrinsic termination. Rho protein functions as a hexamer of a single polypeptide chain with 419 residues, which is the product of the rho gene (5). It is an RNA-binding protein with the capacity to hydrolyze ATP and other nucleoside triphosphates. Rho acts to cause termination by first binding to a site on the nascent transcript and by subsequently using its ATP hydrolysis activity as a source of energy to mediate dissociation of the transcript from RNA polymerase and the DNA template (6). In the cell, the ability of Rho to act at several terminators is dependent upon the presence of an essential 21-kDa protein called NusG (7) that binds both RNA polymerase and Rho itself (8). In vitro the dependence on NusG became apparent only at proximal terminators (at sites ,300 base pairs from the promoter) and under conditions when the RNA molecules are being elongated at the in vivo rate of ;40–50 nucleotides/s (9). The requirement for NusG when RNA chain growth is fast suggests that the NusG is acting to overcome a kinetic limitation of Rho to act alone, perhaps through mediating earlier access to the nascent RNA by the formation of a complex of Rho with RNA polymerase (10). The mechanism of how Rho acts to dissociate the transcription complex is unknown. One important approach to elucidating how interactions with RNA mediate termination of transcription is to determine the structure of the protein. Until a good crystal structure becomes available the properties of its structure will have to be inferred from other, less direct methods, such as biochemical characterization of the protein, phylogenetic comparative analyses, and the functional properties of mutants with known amino acid changes. This review summarizes our current understanding of the structure and function of transcription termination factor Rho based on these indirect approaches.

Loading Rho to terminate transcription

In bacteria, one of the major transcriptional termination mechanisms requires a hexameric RNA/DNA helicase known as Rho. One question that has remained unanswered is how the helicase loads onto a nascent transcript so that it can initiate actions on the transcript to cause termination. Recent structures of Rho bound to nucleic acid by show how the individual RNA-binding domains of the 6 subunits are organized and that the ring is split open. The opening is wide enough to accommodate single-stranded RNA and suggests that this conformation is poised to load onto mRNA.

Initiation of transcription by Escherichia coli RNA polymerase from supercoiled and non-supercoiled bacteriophage PM2 DNA

Abstract Native bacteriophage PM2 DNA, a circular double-helical DNA with —50 superhelical twists, and derivatives of PM2 DNA containing no superhelical twists are compared for their ability to form stable binary complexes with Escherichia coli RNA polymerase. The number of enzyme molecules that can form such complexes, as measured by the number of chains initiated with ATP and GTP in the presence of saturating amounts of enzyme and using heparin to inactivate the unbound enzyme, is 16 per molecule of native PM2 DNA but only about two per molecule of closed non-supercoiled PM2 DNA. In each case equal numbers of chains are initiated with GTP and ATP. The reactivity of the sites on the two forms are also different; the rate of formation and the stability of the complexes are both lower on the non-supercoiled derivatives than on native PM2 DNA. The derivatives also contain a more significant proportion of sites that bind RNA polymerase tightly in an inactive state than native PM2 DNA. Thus, even with excess DNA (so that the number of sites is not limiting) and with sufficient preincubation to form the complexes, the non-supercoiled forms of DNA are still less active templates than native PM2 DNA. On the other hand, the active sites on the two DNAs are similar in one respect; the rates of RNA chain initiation by RNA polymerase bound to these sites are the same.

Attachment of nascent RNA molecules to superhelical DNA

Comparisons are made of the attachment of nascent RNA molecules synthesized in vitro with Escherichia coli RNA polymerase on native bacteriophage PM2 DNA, a double-helical circular DNA with −110 superhelical twists, and on a derivative of this DNA with no superhelical twists. When RNA synthesis is terminated on either DNA by adding EDTA to chelate the Mg2+ ions, the nascent (incompletely polymerized) RNA chains remain bound to the DNA by bonds that involve fewer than 20 nucleotides of the RNA molecule. Differences between the two kinds of DNA are found when the complexes with nascent RNA are treated with sodium dedecyl sulfate or other agents that dissociate proteins (e.g. 3 m -CsCl; 5 m -urea; phenol). With such treatments, all RNA is released from the non-superhelical DNA, whereas only 40% of the RNA molecules are released from the superhelical DNA. The other 60% become bound even more tightly to that DNA, apparently by means of a hybrid helix that can involve up to 600 nucleotides of the bound RNA. It is suggested that the hybrid helix forms spontaneously on the superhelical DNA after denaturing the enzyme because the unwinding of the DNA helix needed to create the RNA-DNA helix relieves some of the strain that is known to exist in the superhelical DNA. Since the extensive hybrid helix does not exist before adding the detergent, the intact structure of the enzyme appears to be important to prevent its formation. Evidently RNA polymerase has a site that dissociates the polymerized portion of the RNA from the DNA as the enzyme moves along the template.

Mechanism of ethidium bromide inhibition of RNA polymerase

Abstract The effect of ethidium bromide on various steps of the reaction catalyzed by Escherichia coli DNA-dependent RNA polymerase is studied. Inhibition caused by low levels (approx. 6 μm) of this DNA-binding drug is a consequence of reducing the rate of RNA chain initiation; the rate of RNA chain growth is unaffected at this concentration. The sensitive step in the initiation process is the formation of stable complexes between RNA polymerase and initiation sites on the DNA. At higher levels (25 μm), ethidium bromide does inhibit the polymerization of those RNA molecules whose initiation has not been blocked.

Identification and characterization of transcription termination sites in the Escherichia coli lacZ gene

The Escherichia coli lacZ gene contains a series of latent transcriptional terminators that are responsible for the polar effects of certain mutations. We demonstrate, using gel electrophoretic size analyses and nuclease S1 mapping procedures, that RNA polymerase terminates RNA synthesis in the vicinity of five positions 180, 220, 379, 421 and 463 base-pairs downstream from the start point during transcription of lacZ DNA in vitro in the presence of rho factor. Termination at all but the 421 position depends on rho factor. In the in vitro assays with 0.05 M-KCl and excess rho (36 nM), the terminators are moderately effective, having efficiencies that range from about 8% at the 180 base-pair site to 56% at the 463 base-pair site. These termination stop points correspond to five of the 11 transcriptional pause sites between 180 and 463 base-pairs. Several stop points also correspond to 3 end points of lacZ mRNA isolated from cells containing the strongly polar lacZ-U118 mutation and from cells starved for serine, thus confirming that these latent terminators are responsible for the polar effect and demonstrating that they also function under a condition of physiological stress that prevents the transcription from being translated properly. Two other potential termination factors, NusA protein and cyclic AMP receptor protein have no effect in vitro on the efficiency of termination at the five lacZ sites.