Absence Of Base Pairing Research Articles

In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson–Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).

The Snakelike Chain Character of Unstructured RNA

The three-dimensional conformation of RNA is governed by a number of physical and chemical factors. However, in the absence of base-pairing and tertiary structure, unstructured RNA assumes a random-walk conformation dominated by the electrostatic self-repulsion of its charged, flexible backbone. This behavior is often modeled as a “wormlike chain” (WLC) with an electrostatics-dependent persistence length. However, through measurements of the end-to-end extension and ion atmosphere of poly(U) RNA stretched by 0.1-10 pN applied force, we find that the WLC model does not adequately describe the behavior of unstructured RNA. Instead, we find that our data are well described by a “snakelike chain” (SLC) model. The SLC model is characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. We see signatures of both regimes in our extension vs. force data: a low-force power-law characteristic of a chain of swollen blobs on long length scales and a high-force region of increasing compliance with ion valence, consistent with increased conformational stabilization, on short length scales. In monovalent salt, we find the crossover between the two regimes (SLC blob size) to scale with the Debye screening length, indicating the determining importance of electrostatics. By systematically varying force and bulk salt concentration, we measure the change in the number of ions associated with the RNA as it is stretched. We find that ions are liberated during stretching. This is consistent with the SLC picture: ions that stabilize the short-length-scale crumples are released when those crumples are mechanically straightened. Additionally, we observe the number of ions liberated increases with increasing bulk salt concentration; this is consistent with the SLC model, but contradicts the WLC model with electrostatics-dependent persistence length.

The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.

Direct Measurements of Base Stacking Interactions in DNA by Single-Molecule Atomic-Force Spectroscopy

We investigate the elasticity of two types of single-stranded synthetic DNA homopolydeoxynucletides, poly(dA) and poly(dT), by AFM-based single-molecule force spectroscopy. We find that poly(dT) exhibits the expected entropic elasticity behavior, while poly(dA) unexpectedly displays two overstretching transitions in the force-extension relationship. We suggest that these transitions, which occur at approximately 23 pN and approximately 113 pN, directly capture, for the first time, the mechanical signature of base-stacking interactions among adenines in DNA, in the absence of base pairing.

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing.

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.

Characterization of U4 and U6 interactions with the 5' splice site using a S. cerevisiae in vitro trans-splicing system.

Spliceosome assembly has been characterized as the ordered association of the snRNP particles U1, U2, and U4/U6.U5 onto pre-mRNA. We have used an in vitro trans-splicing/cross-linking system in Saccharomyces cerevisiae nuclear extracts to examine the first step of this process, 5' splice site recognition. This trans-splicing reaction has ATP, Mg(2+), and splice-site sequence requirements similar to those of cis-splicing reactions. Using this system, we identified and characterized a novel U4-5' splice site interaction that is ATP-dependent, but does not require the branch point, the 3' splice site, or the 5' end of the U1 snRNA. Additionally, we identified several ATP-dependent U6 cross-links at the 5' splice site, indicating that different regions of U6 sample it before a U6-5' splice site interaction is stabilized that persists through the first step of splicing. This work provides evidence for ATP-dependent U4/U6 association with the 5' splice site independent of ATP-mediated U2 association with the branch point. Furthermore, it defines specific nucleotides in U4 and U6 that interact with the 5' splice site at this early stage, even in the absence of base-pairing with the U1 snRNA.

Eukaryotic Nuclear RNase P: Structures and Functions

Over the past decade, ribonucleic acids (RNAs) have gained recognition as the catalysts for an increasing number of biochemical reactions. This chapter discusses the structures and functions of eukaryotic nuclear ribonuclease P (RNase P). The recognition and alignment of appropriate cleavage site proceeds through Watson–Crick base-pairing among the regions of complementary sequence within the same RNA chain. The function of RNase P in vivo is to bind and cleave all of the various transfer RNA (tRNA) precursors. To accomplish this in the absence of base-pairing, common determinants in the three-dimensional structures of the substrates become important. Because of the complex mode of substrate recognition by RNase P, the knowledge of the tertiary structures of both the catalytic and substrate RNAs as well as an understanding of the way these structured RNAs interact is necessary for the determination of its catalytic mechanism. Ribonuclease P is a ribonucleoprotein enzyme that endonucleolytically cleaves precursor-tRNA molecules, generating mature 5' termini. The enzyme RNase P is an essential, well-conserved functionally and has been found in every organism tested. Yeast is a single-cell eukaryotic organism that contains two versions of RNase P: a nuclear form that processes nuclear-encoded pre-tRNAs destined for the cytoplasm and a mitochondrial form that processes pre-tRNAs encoded by the mitochondrial genome.