Biomedical subjects

Simpson Joseph

Publications and source records attributed to Simpson Joseph.

15 recordsLinked to original sources

PubMed2006

A conserved base-pair between tRNA and 23 S rRNA in the peptidyl transferase center is important for peptide release.

The 3' terminus of tRNAs has the universally conserved bases C74C75A76 that interact with the ribosomal large subunit. In the ribosomal P site, bases C74 and C75 of tRNA, form Watson-Crick base-pairs with G2252 and G2251, respectively, present in the conserved P-loop of 23 S rRNA. Previous studies have suggested that the G2252-C74 base-pair is important for peptide bond formation. Using a pure population of mutant ribosomes, we analyzed the precise role of this base-pair in peptide bond formation, elongation factor G-dependent translocation, and peptide release by release factor 1. Surprisingly, our results show that the G2252-C74 base-pair is not essential for peptide bond formation with intact aminoacyl tRNAs as substrates and for EF-G catalyzed translocation. Interestingly, however, peptide release was reduced substantially when base-pair formation between G2252 and C74 of P site tRNA was disrupted, indicating that this conserved base-pair plays an important role in ester bond hydrolysis during translation termination.

Base Pairing↗

PubMed2006

Deletion of a conserved, central ribosomal intersubunit RNA bridge.

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Base Sequence↗

PubMed2006

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.

Base Sequence↗

PubMed2006

Translocation of a tRNA with an extended anticodon through the ribosome.

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

Anticodon↗

PubMed2006

Unfolding of mRNA secondary structure by the bacterial translation initiation complex.

Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.

Codon, Initiator↗

PubMed2006

Ribose 2'-hydroxyl groups in the 5' strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation.

The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA(Met) with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P- and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA(Met) to investigate the contributions of single 2'-hydroxyl groups in the 5' strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2'-hydroxyl groups in the 5' strand of the acceptor arm of P-site-bound tRNA(Met) between positions 1-17 play a critical role during translocation. This suggests that either these 2'-hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

Biological Transport↗

PubMed2005

Simulating movement of tRNA into the ribosome during decoding.

Decoding is the key step during protein synthesis that enables information transfer from RNA to protein, a process critical for the survival of all organisms. We have used large-scale (2.64 x 10(6) atoms) all-atom simulations of the entire ribosome to understand a critical step of decoding. Although the decoding problem has been studied for more than four decades, the rate-limiting step of cognate tRNA selection has only recently been identified. This step, known as accommodation, involves the movement inside the ribosome of the aminoacyl-tRNA from the partially bound "A/T" state to the fully bound "A/A" state. Here, we show that a corridor of 20 universally conserved ribosomal RNA bases interacts with the tRNA during the accommodation movement. Surprisingly, the tRNA is impeded by the A-loop (23S helix 92), instead of enjoying a smooth transition to the A/A state. In particular, universally conserved 23S ribosomal RNA bases U2492, C2556, and C2573 act as a 3D gate, causing the acceptor stem to pause before allowing entrance into the peptidyl transferase center. Our simulations demonstrate that the flexibility of the acceptor stem of the tRNA, in addition to flexibility of the anticodon arm, is essential for tRNA selection. This study serves as a template for simulating conformational changes in large (>10(6) atoms) biological and artificial molecular machines.

Biological Transport↗

PubMed2005

Non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop are essential for ribosomal A site binding and translocation.

The conformation of the anticodon stem-loop of tRNAs required for correct decoding by the ribosome depends on intramolecular and intermolecular interactions that are independent of the tRNA nucleotide sequence. Non-bridging phosphate oxygen atoms have been shown to be critical for the structure and function of several RNAs. However, little is known about the role they play in ribosomal A site binding and translocation of tRNA to the P site. Here, we show that non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop at positions 33, 35, and 37 are important for A site binding. Those at positions 34 and 36 are not necessary for binding, but are essential for translocation. Our results correlate with structural data, indicating that position 34 interacts with the highly conserved 16S rRNA base G966 and position 36 interacts with the universally conserved tRNA base U33 during translocation to the P site.

Anticodon↗

PubMed2005

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit.

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2004

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation.

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.

Protein Biosynthesis↗

PubMed2003

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome.

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.

DNA Primers↗

PubMed2003

After the ribosome structure: how does translocation work?

Structures of the ribosomal large and small subunits have been solved to atomic resolution by X-ray crystallography. These structures provide a new foundation to address the complex process of protein biosynthesis by the ribosome. Translocation of the tRNA-mRNA complex is one of the most fascinating tasks performed by the ribosome. The impact of the crystal structures in understanding the molecular mechanism of translocation is highlighted in this review.

Bacteria↗

PubMed2002

Binding of aminoglycoside antibiotics to the small ribosomal subunit: a continuum electrostatics investigation.

The binding of paromomycin and similar antibiotics to the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from a complex of paromomycin with the 30S subunit was used as a framework to develop structures of similar antibiotics in the same ribosomal binding site. Total binding energies were calculated from electrostatic properties obtained by solution of the Poisson-Boltzmann equation combined with a surface area-dependent apolar term. These computed results showed good correlation with experimental data. Additionally, calculation of the ribosomal electrostatic potential in the paromomycin binding site provided insight into the electrostatic mechanisms for aminoglycoside binding and clues for the rational design of more effective antibiotics.

Anti-Bacterial Agents↗

PubMed2002

Universally conserved interactions between the ribosome and the anticodon stem-loop of A site tRNA important for translocation.

The iterative movement of the tRNA-mRNA complex through the ribosome is a hallmark of the elongation phase of protein synthesis. We used synthetic anticodon stem-loop analogs (ASL) of tRNA(Phe) to systematically identify ribose 2'-hydroxyl groups that are essential for binding and translocation from the ribosomal A site. Our results show that 2'-hydroxyl groups at positions 33, 35, and 36 in the A site ASL are important for translocation. Consistent with the view that the molecular basis of translocation may be similar in all organisms, the 2'-hydroxyl groups at positions 35 and 36 in the ASL interact with universally conserved bases G530 and A1493, respectively, in 16S rRNA. Furthermore, these interactions are also essential for the decoding process, indicating a functional relationship between decoding and translocation.

Anticodon↗

PubMed2002

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.

Base Sequence↗