Simpson Joseph

Electrostatics of nanosystems: Application to microtubules and the ribosome

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme‐substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base‐pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second‐site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild‐type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme‐substrate complex are derived.

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

Translocation, catalyzed by elongation factor EF‐G, is the precise movement of the tRNA–mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A‐ and P‐site tRNAs in EF‐G‐catalyzed translocation by substituting anticodon stem–loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA‐dependent fashion. The requirement for an A‐site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF‐G‐ and GTP‐dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A‐site tRNA are not essential for EF‐G‐dependent translocation. In contrast, no translocation occurs if the P‐site tRNA is substituted with an ASL, indicating that other elements of P‐site tRNA structure are required for translocation. We also tested the effect of increasing the A‐site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon‐D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon‐D arm.

Fragile X Mental Retardation Protein Regulates Translation by Binding Directly to the Ribosome

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. Here, we show that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosome⋅FMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome.

Identification of molecular interactions between P-site tRNA and the ribosome essential for translocation

Translocation of the tRNA–mRNA complex is a fundamental step in the elongation cycle of protein synthesis. Our studies show that the ribosome can translocate a P-site-bound tRNAMet with a break in the phosphodiester backbone between positions 56 and 57 in the TΨC-loop. We have used this fragmented P-site-bound tRNAMet to identify two 2′-hydroxyl groups at positions 71 and 76 in the 3′-acceptor arm that are essential for translocation. Crystallographic data show that the 2′-hydroxyl group at positions 71 and 76 contacts the backbone of 23S rRNA residues 1892 and 2433–2434, respectively, in the ribosomal E site. These results establish a set of functional interactions between P-site tRNA and 23S rRNA that are essential for translocation.

Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome.

In order to map the rRNA environment of the acceptor end of tRNA in th e ribosome, hydroxyl radicals were generated in situ from Fe(II) attached via an EDTA linker to the 5′ end of tRNA. Nucleotides in rRNA cleaved by the radicals were identified by primer extension, and assigned to the ribosomal A, P and E sites by standard criteria. In the A site, cleavages were found in the 2555–2573 region of 23S rRNA, around bases previously shown to be protected by A site tRNA, and in the alpha‐sarcin loop, the site of interaction of elongation factors EF‐Tu and EF‐G. P site cleavages occurred in the 2250 loop, where a base pair is made with C74 of tRNA; and around the 2493 region in domain V. Interestingly, two clusters of nucleotides in 23S rRNA are accessible to both A site and P site tRNA probes. The first cluster is in the 1940–1965 region of domain IV, around the site of affinity labeling by the 3′ end of tRNA, and the second cluster is around the bulged adenosine A2602, whose accessibility to chemical probes is enhanced by P site tRNA and decreased by A site tRNA. From the E site, cleavages occur in the 2390–2440 region, surrounding C2394, a base protected from dimethyl sulfate by E site tRNA, and in the phylogenetically variable stem at positions 1860/1880 of domain IV. Unexpectedly, no cleavages were detected in the central loop of domain V of 23S rRNA.

Understanding Discrimination by the Ribosome: Stability Testing and Groove Measurement of Codon- Anticodon Pairs

The ribosome must discriminate between correct and incorrect tRNAs with sufficient speed and accuracy to sustain an adequate rate of cell growth. Here, we report the results of explicit solvent molecular dynamics simulations, which address the mechanism of discrimination by the ribosome. The universally conserved 16S rRNA base A1493 and the kink in mRNA between A and P sites amplify differences in stability between cognate and near-cognate codon-anticodon pairs. Destabilization by the mRNA kink also provides a geometric explanation for the higher error rates observed for mismatches in the first codon position relative to mismatches in the second codon position. For more stable near-cognates, the repositioning of the universally conserved bases A1492 and G530 results in increased solvent exposure and an uncompensated loss of hydrogen bonds, preventing correct codon-anticodon-ribosome interactions from forming.

The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

Helix 38 (H38) in 23 S rRNA, which is known as the “A-site finger (ASF),” is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

All-atom homology model of the Escherichia coli 30S ribosomal subunit

Understanding the structural basis of ribosomal function requires close comparison between biochemical and structural data. Although a large amount of biochemical data are available for the Escherichia coli ribosome, the structure has not been solved to atomic resolution. Using a new RNA homology procedure, we have modeled the all-atom structure of the E. coli 30S ribosomal subunit. We find that the tertiary structure of the ribosome core, including the A-, P- and E-sites, is highly conserved. The hypervariable regions in our structure, which differ from the structure of the 30S ribosomal subunit from Thermus thermophilus, are consistent with the cryo-EM map of the E. coli ribosome.

Functional Role of the Sarcin-Ricin Loop of the 23S rRNA in the Elongation Cycle of Protein Synthesis

The sarcin-ricin loop (SRL) is one of the longest conserved sequences in the 23S ribosomal RNA. The SRL has been accepted as crucial for the activity of the ribosome because it is targeted by cytotoxins such as α-sarcin and ricin that completely abolish translation. Nevertheless, the precise functional role of the SRL in translation is not known. Recent biochemical and structural studies indicate that the SRL is critical for triggering GTP hydrolysis on elongation factor Tu (EF-Tu) and elongation factor G (EF-G). To determine the functional role of the SRL in the elongation stage of protein synthesis, we analyzed mutations in the SRL that are known to abolish protein synthesis and are lethal to cells. Here, we show that the SRL is not critical for GTP hydrolysis on EF-Tu and EF-G. The SRL also is not essential for peptide bond formation. Our results, instead, suggest that the SRL is crucial for anchoring EF-G on the ribosome during mRNA-tRNA translocation.