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1.
Methods Mol Biol ; 969: 55-72, 2013.
Article in English | MEDLINE | ID: mdl-23296927

ABSTRACT

The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.


Subject(s)
Protein Biosynthesis , RNA Cap Analogs/chemistry , RNA Stability , RNA, Messenger , Transfection/methods , Animals , Humans , RNA, Messenger/chemical synthesis , RNA, Messenger/chemistry , RNA, Messenger/genetics
2.
RNA ; 19(1): 1-16, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23188809

ABSTRACT

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem-loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5'→3' and 3'→5' processes, but the relative contributions are not known. The 3' end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3' UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5'→3' decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5' decay intermediates were oligouridylated. Preventing oligouridylation by 3'-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3'→5' degradation machinery stalls during initial degradation, whereupon reuridylation occurs.


Subject(s)
Oligoribonucleotides/metabolism , RNA Stability/physiology , RNA, Messenger/metabolism , Uracil Nucleotides/metabolism , 3' Untranslated Regions/physiology , DNA Replication/drug effects , Deoxyadenosines/pharmacology , Gene Silencing , HeLa Cells , Histones/metabolism , Humans , Hydroxyurea/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Oligoribonucleotides/antagonists & inhibitors , Polynucleotide Adenylyltransferase , Protein Biosynthesis , RNA Stability/genetics , RNA, Messenger/chemistry , Transduction, Genetic , Uracil Nucleotides/antagonists & inhibitors , mRNA Cleavage and Polyadenylation Factors
3.
J Biol Chem ; 283(37): 25227-25237, 2008 Sep 12.
Article in English | MEDLINE | ID: mdl-18614538

ABSTRACT

Interaction of the mRNA cap with the translational machinery is a critical and early step in the initiation of protein synthesis. To better understand this process, we determined kinetic constants for the interaction of m(7)GpppG with human eIF4E by stopped-flow fluorescence quenching in the presence of a 90-amino acid fragment of human eIF4G that contains the eIF4E-binding domain (eIF4G(557-646)). The values obtained, k(on) = 179 x 10(6) m(-1) s(-1) and k(off) = 79 s(-1), were the same as reported previously in the absence of an eIF4G-derived peptide. We also used surface plasmon resonance to determine kinetic constants for the binding of eIF4E to eIF4G(557-646), both in the presence and absence of m(7)GpppG. The results indicated that eIF4G(557-646) binds eIF4E and eIF4E.m(7)GpppG at the same rate, with k(on) = 3 x 10(6) m(-1) s(-1) and k(off) = 0.01 s(-1). Our data represent the first full kinetic description of the interaction of eIF4E with its two specific ligands. The results demonstrate that the formation of the m(7)GpppG.eIF4E.eIF4G(557-646) complex obeys a sequential, random kinetic mechanism and that there is no preferential pathway for its formation. Thus, even though eIF4G(557-646) binds eIF4E tightly, it does not increase the affinity of eIF4E for m(7)GpppG, as has been claimed in several previous publications. We did, in fact, observe increased binding to m(7)GTP-Sepharose in the presence of eIF4G(557-646), but only with recombinant eIF4E that was prepared from inclusion bodies.


Subject(s)
Eukaryotic Initiation Factor-4E/chemistry , Eukaryotic Initiation Factor-4G/chemistry , RNA Cap Analogs/chemistry , RNA Caps/chemistry , Amino Acid Sequence , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Humans , Kinetics , Ligands , Microscopy, Fluorescence , Molecular Sequence Data , Protein Biosynthesis , Saccharomyces cerevisiae/metabolism , Sepharose/chemistry , Sequence Homology, Amino Acid , Surface Plasmon Resonance
4.
J Biol Chem ; 281(21): 14927-38, 2006 May 26.
Article in English | MEDLINE | ID: mdl-16540463

ABSTRACT

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.


Subject(s)
Eukaryotic Initiation Factor-4E/chemistry , Oligoribonucleotides/chemistry , Animals , Humans , Kinetics , Mice , Models, Chemical , Molecular Conformation , Oligonucleotides/chemistry , Phosphorylation , Potassium Chloride/chemistry , Protein Binding , Recombinant Proteins/chemistry
5.
FEBS Lett ; 565(1-3): 65-9, 2004 May 07.
Article in English | MEDLINE | ID: mdl-15135054

ABSTRACT

Recent reports have indicated that insect antimicrobial peptides kill bacteria by inhibiting the molecular chaperone DnaK. It was proposed that the antimicrobial peptide, all-L-pyrrhocoricin (L-PYR), binds to two sites on DnaK, the conventional substrate-binding site and the multi-helical C-terminal lid, and that inhibition of DnaK comes about from the lid mode of binding. In this report, we show using two different assays that L-PYR binds to and stimulates the ATPase activity of both wild-type and a lidless variant of DnaK. Our study shows that L-PYR interacts with DnaK much like the all-L NR (NRLLLTG) peptide, which is known to bind in the conventional substrate-binding site of DnaK. L-PYR antimicrobial activity is thus a consequence of the competitive inhibition of bacterial DnaK.


Subject(s)
Adenosine Triphosphatases/chemistry , Anti-Bacterial Agents/chemistry , Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Insect Proteins , Animals , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Binding Sites , Dose-Response Relationship, Drug , HSP70 Heat-Shock Proteins/metabolism , Insecta , Kinetics , Models, Chemical , Models, Molecular , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Spectrometry, Fluorescence , Temperature , Time Factors
6.
Biochemistry ; 42(30): 9028-40, 2003 Aug 05.
Article in English | MEDLINE | ID: mdl-12885236

ABSTRACT

In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor. Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE. We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude. SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted. Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK. This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid. The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed. Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE. We discuss the impact of each domain on complex formation and dissociation.


Subject(s)
Bacterial Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Sequence Deletion , Thermodynamics , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/metabolism , Catalysis , Colorimetry/methods , Escherichia coli Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Kinetics , Macromolecular Substances , Mutagenesis, Site-Directed , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Substrate Specificity/genetics , Surface Plasmon Resonance/methods
7.
Biochemistry ; 42(19): 5867-76, 2003 May 20.
Article in English | MEDLINE | ID: mdl-12741845

ABSTRACT

The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region is not known. By sequentially deleting the flexible subdomain and the individual lid helices, we deduced the importance of each structural unit to creating long-lived DnaK-peptide complexes. Here we report that (i) the alphaD helix is essential for long-lived DnaK-peptide complexes. For example, ATP triggers the dissociation of a acrylodan-labeled p5 peptide (ap5, a-CLLLSAPRR) from wtDnaK and DnaK595(A-D) with k(off) equal to 7.6 and 8.9 s(-1), respectively, whereas when the D-helix is deleted, creating DnaK578(A-C), k(off) jumps to 207 s(-1). (ii) The presence of the alphaB helix impacts the rate of the ATP-induced high-to-low affinity conformational change. For example, ATP induces this conformational change in a lidless variant, DnaK517(1/2A), with a rate constant of 442 s(-1), whereas, after adding back the B-helix (residues 518-554), ATP induces this conformational change in DnaK554(A-B) with a rate constant of 2.5 s(-1). Our interpretation is that this large decrease occurs because the B-helix of the DnaK554(A-B) is bound in the substrate-binding site. (iii) The deletion analysis also revealed that residues 596-638, which comprise the alphaE helix and the flexible subdomain, affect ATP binding. Our results are consistent with this part of the lid producing conformational heterogeneity, perhaps by binding to the ATPase domain.


Subject(s)
Adenosine Triphosphate/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Kinetics , Models, Molecular , Molecular Chaperones/genetics , Mutagenesis, Site-Directed , Plasmids/genetics , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Deletion , Spectrometry, Fluorescence , Tryptophan/chemistry
8.
FEBS Lett ; 539(1-3): 100-4, 2003 Mar 27.
Article in English | MEDLINE | ID: mdl-12650934

ABSTRACT

The molecular chaperone DnaK is composed of two functional domains, the ATPase domain and the substrate-binding domain. In this report, we show that peptide binding to DnaK can be sensed in real time through a labeled nucleotide bound in the ATPase domain. Specifically, when N8-(4-N'-methylanthraniloylaminobutyl)-8-aminoadenosine 5'-triphosphate (MABA)-ATP.DnaK complexes are rapidly mixed with excess peptide, MABA fluorescence rapidly increases and the rate of increase is proportional to peptide concentration. Analysis of the formation traces yield on and off rate constants that are exactly equal to the rate constants obtained from experiments that directly probe peptide binding to DnaK. These results are the first to show that peptide binding to ATP.DnaK triggers a concerted conformational change in the ATPase domain.


Subject(s)
Escherichia coli Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Adenosine Triphosphatases/metabolism , Escherichia coli Proteins/chemistry , HSP70 Heat-Shock Proteins/chemistry , Peptides/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary
9.
Biochemistry ; 41(40): 12224-35, 2002 Oct 08.
Article in English | MEDLINE | ID: mdl-12356325

ABSTRACT

DnaK, the Escherichia coli Hsp70, possesses two functional domains, the N- and C-terminal ATPase and peptide-binding domains, respectively. Elucidation of the mechanism of allosteric coupling between the two domains is key to understanding how Hsp70 chaperones interact with their substrates. We previously reported that ATP reacts with wild-type DnaK-peptide complexes according to the two-step reaction, ATP + DnaK-P if ATP-DnaK-P if ATP-DnaK + P, where ATP binds in the first step, and a conformational change that quenches DnaK's tryptophan fluorescence (denoted by the asterisk) and expels bound peptide occurs in the second step. Here we report that DnaK(2-517), a lidless variant, also reacts with ATP and peptide by this two-step mechanism. Compared to wild-type DnaK, we found that, depending on the sequence of the bound peptide and the temperature, deletion of the lid produces a 27- to 66-fold increase in the rate constant (k(2)) for the ATP-triggered conformational change (ATP-DnaK-P --> ATP-DnaK+P) but only a approximately 2-fold increase in the rate constant (k(-)(2)) for the reverse reaction (ATP-DnaK+P --> ATP-DnaK-P). A model is proposed in which the lid regulates the rate of interdomain communication by retarding motions within the beta-sandwich that occur as a consequence of ATP binding. New evidence in support of the reversible, two-step conformational switch mechanism is also presented.


Subject(s)
2-Naphthylamine/analogs & derivatives , Escherichia coli Proteins , HSP70 Heat-Shock Proteins/chemistry , Molecular Chaperones/chemistry , 2-Naphthylamine/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Fluorescent Dyes/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Kinetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Protein Structure, Tertiary , Sequence Deletion , Spectrometry, Fluorescence , Tryptophan/metabolism
10.
Mol Microbiol ; 45(5): 1197-206, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12207689

ABSTRACT

We discuss recent experiments that have illuminated individual steps in the reaction cycle of the Escherichia coli Hsp70 molecular chaperone DnaK. Using this new information, we compare two distinctly different global mechanisms of action--holding versus unfolding--and argue that the available evidence suggests that DnaK is an unfoldase.


Subject(s)
Escherichia coli Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Adenosine Triphosphate/metabolism , Computer Simulation , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , HSP70 Heat-Shock Proteins/chemistry , Models, Biological , Models, Molecular , Molecular Chaperones/chemistry , Protein Biosynthesis , Protein Folding , Protein Processing, Post-Translational , Protein Structure, Tertiary
11.
Biochemistry ; 41(26): 8499-507, 2002 Jul 02.
Article in English | MEDLINE | ID: mdl-12081501

ABSTRACT

In this study, the effect of pH on the conformation and the reactivity of the Escherichia coli Hsp70 molecular chaperone DnaK was investigated using spectroscopic and chemical assays. DnaK exhibits negligible binding of the hydrophobic dye 1-anilino-naphthalene-8-sulfonate (ANS) between pH 7 to 5.0, whereas appreciable binding occurs between pH 4.5 to 4.0. The binding of ANS to a protein is diagnostic of the presence of accessible ordered hydrophobic surfaces. Such hydrophobic surfaces are often displayed by partially folded protein intermediates such as molten globules. Nucleotide inhibits 70% of the ANS binding at pH 4.5 but none of the ANS binding at pH 4.0. Proteolysis of nucleotide-free DnaK at pH 4.5 with cathepsin D yields detectable fragments (masses > 20 kDa) of the C-terminal peptide-binding domain but none of the N-terminal ATPase domain, thus the ATPase domain is preferentially targeted for proteolysis. In contrast, proteolysis of nucleotide-free DnaK at pH 4.0 with cathepsin D cuts near the linker region, yielding both functional domains. Our interpretation of these data is that incubation of DnaK at pH 4.5 produces a partially unfolded form of the ATPase domain, in which secondary structure is mainly intact, but tertiary structure is reduced. Incubation of the protein at pH 4.0 produces an intermediate in which both functional domains have collapsed and possibly separated. Nucleotide inhibits the conformational change that occurs at pH 4.5 but not at 4.0.


Subject(s)
Escherichia coli Proteins , HSP70 Heat-Shock Proteins/metabolism , Hydrogen-Ion Concentration , Molecular Chaperones/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Calorimetry , Circular Dichroism , Escherichia coli/metabolism , Fluorescent Dyes , HSP70 Heat-Shock Proteins/chemistry , Kinetics , Models, Molecular , Molecular Chaperones/chemistry , Protein Conformation , Protein Denaturation , Thermodynamics
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