Effect of 8-Br-ATP and 8-Oxy-ATP on RNA synthesis by RNA polymerase from Escherichia coli

The effect of 8-Br-ATP and 8-oxy-ATP on RNA synthesis on calf thymus DNA and on abortive synthesis of di- and trinucleotides on promoter AI of phage T7 delta DIII DNA in the case of an incomplete set of substrates was studied. It was shown that the ATP analogs used inhibit the RNA and di- and trinucleotide synthesis. In all cases, 8-oxy-ATP was a more effective inhibitor than 8-Br-ATP. Both analogs are incapable of being the primer and they do not replace ATP in the course of abortive initiation of pppApU synthesis.     

Intracellular Succinylation of 8-Chloroadenosine and Its Effect on Fumarate Levels

8-Chloroadenosine (8-Cl-Ado) is a ribosyl nucleoside analog currently in phase I testing for the treatment of chronic lymphocytic leukemia (CLL). 8-Cl-Ado activity is dependent on adenosine kinase and requires intracellular accumulation of 8-Cl-Ado as mono-, di-, and tri-phosphates. In the current study with four mantle cell lymphoma cell lines, we report a new major metabolic pathway for 8-Cl-Ado intracellular metabolism, the formation of succinyl-8-chloro-adenosine (S-8-Cl-Ado) and its monophosphate (S-8-Cl-AMP). 8-Cl-AMP levels were highly associated with S-8-Cl-AMP levels and reached a steady-state prior to the secondary metabolites, 8-Cl-ATP and S-8-Cl-Ado. Consistent with fumarate as a required substrate for formation of succinyl-8-Cl-adenylate metabolites, the S-8-Cl-adenylate concentrations in multiple cell lines were associated with fumarate loss. The distribution of metabolites was also altered using the energy metabolism modifiers, metformin and oligomycin. The rates of succinyl-8-Cl-adenylate metabolism were enhanced by increasing the intracellular fumarate concentrations after metformin co-treatment. In addition, the S-8-Cl-AMP concentrations were increased after acute inhibition of ATP synthase by oligomycin. We conclude that 8-Cl-Ado metabolism not only affects intracellular purine metabolism; 8-Cl-Ado conversion to succinyl analogs ties its metabolism to the citric acid cycle by reduction of the fumarate pool.     

Multiple sequence elements and a maternal mRNA product control cdk2 RNA polyadenylation and translation during early Xenopus development.

Cytoplasmic poly(A) elongation is one mechanism that regulates translational recruitment of maternal mRNA in early development. In Xenopus laevis, poly(A) elongation is controlled by two cis elements in the 3' untranslated regions of responsive mRNAs: the hexanucleotide AAUAAA and a U-rich structure with the general sequence UUUUUAAU, which is referred to as the cytoplasmic polyadenylation element (CPE). B4 RNA, which contains these sequences, is polyadenylated during oocyte maturation and maintains a poly(A) tail in early embryos. However, cdk2 RNA, which also contains these sequences, is polyadenylated during maturation but deadenylated after fertilization. This suggests that cis-acting elements in cdk2 RNA signal the removal of the poly(A) tail at this time. By using poly(A) RNA-injected eggs, we showed that two elements which reside 5' of the CPE and 3' of the hexanucleotide act synergistically to promote embryonic deadenylation of this RNA. When an identical RNA lacking a poly(A) tail was injected, these sequences also prevented poly(A) addition. When fused to CAT RNA, the cdk2 3' untranslated region, which contains these elements, as well as the CPE and the hexanucleotide, promoted poly(A) addition and enhanced chloramphenicol acetyltransferase activity during maturation, as well as repression of these events after fertilization. Incubation of fertilized eggs with cycloheximide prevented the embryonic inhibition of cdk2 RNA polyadenylation but did not affect the robust polyadenylation of B4 RNA. This suggests that a maternal mRNA, whose translation occurs only after fertilization, is necessary for the cdk2 deadenylation or inhibition of RNA polyadenylation. This was further suggested when poly(A)+ RNA isolated from two-cell embryos was injected into oocytes that were then allowed to mature. Such oocytes became deficient for cdk2 RNA polyadenylation but remained proficient for B4 RNA polyadenylation. These data show that CPE function is developmentally regulated by multiple sequences and factors.     

Assembly of a processive messenger RNA polyadenylation complex.

Polyadenylation of mRNA precursors by poly(A) polymerase depends on two specificity factors and their recognition sequences. These are cleavage and polyadenylation specificity factor (CPSF), recognizing the polyadenylation signal AAUAAA, and poly(A) binding protein II (PAB II), interacting with the growing poly(A) tail. Their effects are independent of ATP and an RNA 5'-cap. Analysis of RNA-protein interactions by non-denaturing gel electrophoresis shows that CPSF, PAB II and poly(A) polymerase form a quaternary complex with the substrate RNA that transiently stabilizes the binding of poly(A) polymerase to the RNA 3'-end. Only the complex formed from all three proteins is competent for the processive synthesis of a full-length poly(A) tail.     

In vitro cleavage of the simian virus 40 early polyadenylation site adjacent to a required downstream TG sequence.

Exogenous RNA containing the simian virus 40 early polyadenylation site was efficiently and accurately polyadenylated in in vitro nuclear extracts. Correct cleavage required ATP. In the absence of ATP, nonpoly(A)+ products accumulated which were 18 to 20 nucleotides longer than the RNA generated by correct cleavage; the longer RNA terminated adjacent to the downstream TG element required for polyadenylation. In the presence of ATP analogs, alternate cleavage was not observed; instead, correct cleavage without poly(A) addition occurred. ATP-independent cleavage of simian virus 40 early RNA had many of the same properties as correct cleavage including requirements for an intact AAUAAA element, a proximal 3' terminus, and extract small nuclear ribonucleoproteins. This similarity in reaction parameters suggested that ATP-independent cleavage is an activity of the normal polyadenylation machinery. The ATP-independent cleavage product, however, did not behave as an intermediate in polyadenylation. The alternate RNA did not preferentially chase into correctly cleaved material upon readdition of ATP; instead, poly(A) was added to the 3' terminus of the cleaved RNA during a chase. Purified ATP-independent cleavage RNA, however, was a substrate for correct cleavage when reintroduced into the nuclear extract. Thus, alternate cleavage of polyadenylation sites adjacent to a required downstream sequence element is directed by the polyadenylation machinery in the absence of ATP.     

The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3-4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3'-terminal adenosines and, over several adenylation steps, elicits precisely tuned adjustments of ATP affinities and rate constants for adenylation and TRAMP dissociation. Our data establish Mtr4p as a critical regulator of polyadenylation by TRAMP and reveal that an RNA helicase can control the activity of another enzyme in a highly complex fashion and in response to features in RNA.     

Inhibition of the polyadenylation reaction in vitro by polyamines

The effect of polyamines on the polyadenylation reaction in vitro was investigated. Varying concentrations of spermine were added to the reaction catalyzed by purified poly(A) polymerase using rat liver nuclear RNA, poly(A), Escherichia coli tRNA or (Ap)₃A as exogenous primers. The enzyme activity decreased progressively with increasing concentrations of polyamines; complete inhibition was obtained at 0.4 and 1.2 mm spermine for the nuclear RNA- and poly(A)-primed reactions, respectively. No inhibition was observed for the (Ap)₃A-primed reaction. Spermidine and putrescine also inhibited polyadenylation but to a lesser extent than spermine. The degree of inhibition by spermine was related to the polynucleotide primer concentrations. Spermine prevented polyadenylation by binding to the primer but not to the poly(A) polymerase molecule as shown by the migration of [¹⁴C]spermine through glycerol gradients after preincubation with enzyme or tRNA. At concentrations inhibitory to polyadenylation in vitro, spermine could stimulate the DNA-dependent RNA synthesis catalyzed by RNA polymerase II. The present study suggests that low levels of polyamines could be used as specific inhibitors of the poly(A) synthesis in vitro.     

Uridylate-containing RNA sequences determine specificity for binding and polyadenylation by the catalytic subunit of vaccinia virus poly(A) polymerase.

VP55, the catalytic subunit of vaccinia virus poly(A) polymerase, has the remarkable property of adding 30-35 adenylates to RNA 3' ends in a rapid processive burst before an abrupt transition to slow, non-processive adenylate addition. Here, we demonstrate that this property results from the affinity of the enzyme for uridylate residues within the 3' 31-40 nt of the RNA primer. At physiological salt concentrations, both polyadenylation and stable VP55 binding required the presence of multiple uridylates within a 31-40 nt length of RNA, though specific RNA sequences were not necessary. Even DNA in which the deoxythymidylate residues were replaced with ribouridylates, could be polyadenylated in a processive manner. Both the unmethylated pyrimidine ring and a 2'-OH on the associated sugar are features of ribouridylates that are important for priming. The abrupt termination of processive polyadenylation was attributed to translocation of VP55 along the nascent poly(A) tail, which lacks uridylates for stable binding. As evidence for translocation and interaction with newly synthesized RNA, other homopolymer tails were synthesized by VP55 in the presence of Mn2+, which relaxes its donor nucleotide specificity. Only during poly(U) tail synthesis did processive nucleotide addition fail to terminate.     

Covalent modification of Escherichia coli ADPglucose synthetase with 8-azido substrate analogs

Two photoaffinity labeling agents, 8-azido-ATP and 8-azido-ADPglucose, are substrate site specific probes of the Escherichia coli ADPglucose synthetase. In the presence of light (254 nm), the analogs specifically and covalently modify the enzyme with concomitant loss of catalytic activity. The substrate ADPglucose completely protects the enzyme from covalent modification by these 8-azido analogs. ATP, another substrate, also provides nearly 100% protection from 8-azido-ATP inactivation but is less efficient in protection of inactivation by 8-azido-ADPglucose. In the absence of light, however, ADPglucose synthetase can utilize either 8-azido-ATP or 8-azido-ADPglucose as substrates.     

Secondary structure of the hybrid poly(rA).poly(dT) in solution. Studies involving NOE at 500 MHz and stereochemical modelling within the constraints of NOE data

One-dimensional nuclear Overhauser effect (NOE) in nuclear magnetic resonance spectroscopy along with stereochemically sound model building was employed to derive the structure of the hybrid poly(rA).poly(dT) in solution. Extremely strong NOE was observed at AH2' when AH8 was presaturated; strong NOEs were observed at TH2'TH2' when TH6 was presaturated; in addition the observed NOEs at TH2' and TH2' were nearly equal when TH6 was presaturated. There was no NOE transfer to AH3' from AH8 ruling out the possibility of (C-3'-endo, low anti chi approximately equal to 200 degrees to 220 degrees) conformation for the A residues. The observed NOE data suggest that the nucleotidyl units in both rA and dT strands have equivalent conformations: C-2'-endo/C-1'-exo, anti chi approximately equal to 240 degrees to 260 degrees. Such a nucleotide geometry for rA/dT is consistent with a right-handed B-DNA model for poly(rA).poly(dT) in solution in which the rA and dT strands are conformationally equivalent. Molecular models were generated for poly(rA).poly(dT) in the B-form based upon the geometrical constraints as obtained from the NOE data. Incorporation of (C-2'-endo pucker, chi congruent to 240 degrees to 260 degrees) into the classical B-form resulted in severe close contacts in the rA chain. By introducing base-displacement, tilt and twist along with concomitant changes in the backbone torsion angles, we were able to generate a B-form for the hybrid poly(rA).poly(dT) fully consistent with the observed NOE data. In the derived model the sugar pucker is C-1'-exo, a minor variant of C-2'-endo and the sugar base torsion is 243 degrees, the remaining torsion angles being: epsilon = 198 degrees, xi = 260 degrees, alpha = 286 degrees, beta = 161 degrees and gamma = 72 degrees; this structure is free of any steric compression and indicates that it is not necessary to switch to C-3'-endo pucker for rA residues in order to accommodate the 2'-OH group. The structure that we have proposed for the polynucleotide RNA-DNA hybrid in solution is in complete agreement with that proposed for a hexamer hybrid in solution from NOE data and is inconsistent with the heteronomous model proposed for the fibrous state.     

RNA processing in vitro produces mature 3'' ends of a variety of Saccharomyces cerevisiae mRNAs.

Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.     

Primer specificity of ribosome-associated poly(A) polymerase from Ehrlich ascites tumour cells

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA nucleotidyltransferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8--30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12--18 adenylate residues to pre-existing mRNA poly(A) sequences of 40--160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA. We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.     

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA polyadenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3′-ends of most of the mRNA molecules in bacteria are characterized by a stem–loop structure. We show here that in vitro PAP I activity is inhibited by a stem–loop structure. A tail of two to six nucleotides located 3′ to the stem–loop structure is sufficient to overcome this inhibition. These results suggest that the stem–loop structure located in most of the mRNA 3′-ends may function as an inhibitor of polyadenylation and degradation of the corresponding RNA molecule. However, RNA 3′-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.     

Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.     

2'-Deoxy-2'-azidoadenosine triphosphate and 2'-deoxy-2'-fluoroadenosine triphosphate as substrates and inhibitors for Escherichia coli DNA-dependent RNA polymerase

The effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AZTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.