Interaction between the Spliceosomal Pre-mRNA Branch Site and U2 snRNP Protein p14.

We have probed the molecular basis of recognition between human spliceosomal U2 snRNP protein p14 and RNA targets representing the intron branch site region. Interaction of an RNA duplex representing the branch site helix perturbed at least 10 nuclear magnetic resonance cross-peaks of (15)N-labeled p14. However, similar chemical shift changes were observed upon interaction with a duplex without the bulged branch site residue, suggesting that binding of p14 to RNA is nonspecific and does not recognize the branch site. We propose that the p14-RNA interaction screens charges on the backbone of the branch site during spliceosome assembly.

Structure and folding of a rare, natural kink turn in RNA with an A*A pair at the 2b*2n position.

The kink turn (k-turn) is a frequently occurring motif, comprising a bulge followed by G•A and A•G pairs that introduces a sharp axial bend in duplex RNA. Natural k-turn sequences exhibit significant departures from the consensus, including the A•G pairs that form critical interactions stabilizing the core of the structure. Kt-23 found in the small ribosomal subunit differs from the consensus in many organisms, particularly in the second A•G pair distal to the bulge (2b•2n). Analysis of many Kt-23 sequences shows that the frequency of occurrence at the 2n position (i.e., on the nonbulged strand, normally G in standard k-turns) is U>C>G>A. Less than 1% of sequences have A at the 2n position, but one such example occurs in Thelohania solenopsae Kt-23. This sequence folds only weakly in the presence of Mg²âº ions but is induced to fold normally by the binding of L7Ae protein. Introduction of this sequence into the SAM-I riboswitch resulted in normal binding of SAM ligand, indicating that tertiary RNA contacts have resulted in k-turn folding. X-ray crystallography shows that the T. solenopsae Kt-23 adopts a standard k-turn geometry, making the key, conserved hydrogen bonds in the core and orienting the 1n (of the bulge-proximal A•G pair) and 2b adenine nucleobases in position facing the opposing minor groove. The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands.

Impact of base pair identity 5' to the spliceosomal branch site adenosine on branch site conformation.

The branch site helix from Saccharomyces cerevisiae with pseudouridine (ψ) incorporated in a phylogenetically conserved position of U2 snRNA features an extrahelical branch site adenosine (A) that forms a base triple interaction with the minor groove edge of a widely conserved purine(U2 strand)-pyrimidine(intron strand) (R(U2)-Y(intron)) base pair two positions upstream. In these studies, NMR spectra of a duplex in which 2-aminopurine (2ap), a fluorescent analog of adenine lacking the proposed hydrogen bond donor, was substituted for the branch site A, indicated that the substitution does not alter the extrahelical position of the branch site residue; thus, it appears that a hydrogen bond between the adenine amino group and the R-Y pair is not obligatory for stabilization of the extrahelical conformation. In contrast, reversal of the orientation of A(U2)-U(intron) to U(U2)-A(intron) resulted in an intrahelical position for the branch site A or 2ap. Fluorescence intensity of 2ap substituted for the branch site A with the original R(U2)-Y(intron) orientation (AU or GC) was high, consistent with an extrahelical position, whereas fluorescence in helices with the reversed R-Y orientation, or with a mismatched pair (A-U → G•A or U•C), was markedly quenched, implying that the residue was stacked in the helix. The A 5' to the branch site residue was not extrahelical in any of the duplexes. These findings suggest that the R(U2)-Y(intron) base pair orientation in the ψ-dependent branch site helix plays an important role in positioning the branch site A for recognition and/or function.

Methods of biotechnology Biotech BioBrawl: A competition-based learning approach to biotechnology.

Biotechnology students entering the workforce often struggle in their application of textbook knowledge to build the solutions that we see in science and health fields today. Some students may be naive to what a job in the biotechnology industry can encompass. Students should graduate having a firm grasp of the prospects of their field and have the confidence to begin contributing to the growth of the industry. For this, it is necessary for students to be able to start practising applications in their coursework before they graduate. A competition titled "Biotech BioBrawl" was incorporated in the University of Central Florida's Methods in Biotechnology (MCB4721C/MCB5722C) course agenda during the semester of Fall 2021. This competition challenged students to harness innovation and applied science in a group setting that led to the development and pitch of an original idea to a panel of judges with various biotechnology industry experiences.

Single-molecule observation of the induction of k-turn RNA structure on binding L7Ae protein.

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.

A structural database for k-turn motifs in RNA.

The kink-turn (k-turn) is a common structural motif in RNA that introduces a tight kink into the helical axis. k-turns play an important architectural role in RNA structures and serve as binding sites for a number of proteins. We have created a database of known and postulated k-turn sequences and three-dimensional (3D) structures, available via the internet. This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known.

Ion-induced folding of a kink turn that departs from the conventional sequence.

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G*A and A*G base pairs, and when these G*A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A*G pair is replaced by a non-Watson-Crick A*U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg(2+) or Na(+) ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A*G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson-Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A*U pair is more important than the other.

Applying active learning in a virtual classroom such as a molecular biology escape room.

The transition to remote-teaching online for Molecular Biology has forced active learning exercises like Escape Rooms to also move online. In the past, Escape Rooms have been an effective tool for students to help reinforce concepts they learned in Molecular Biology. We propose that there is a way for Escape Rooms to be moved to an online setting and still be an effective avenue for students to learn the material in a fun and interactive way.

A case-based learning approach to online biochemistry labs during COVID-19.

With biochemistry forced to transition to remote-teaching online, the cooperative active learning and problem-solving normally in labs have been limited. With little ability to perform experiments with laboratory equipment, determining how to mimic the qualities integral to these labs in an online environment is necessary. We propose one possible solution to provide online labs: short case-based learning activities.

RNA tertiary interactions in a riboswitch stabilize the structure of a kink turn.

The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.

NMR spectroscopy of RNA duplexes containing pseudouridine in supercooled water.

We have performed NMR experiments in supercooled water in order to decrease the temperature-dependent exchange of protons in RNA duplexes. NMR spectra of aqueous samples of RNA in bundles of narrow capillaries that were acquired at temperatures as low as -18 degrees C reveal resonances of exchangeable protons not seen at higher temperatures. In particular, we detected the imino protons of terminal base pairs and the imino proton of a non-base-paired pseudouridine in a duplex representing the eukaryotic pre-mRNA branch site helix. Analysis of the temperature dependence of chemical shift changes (thermal coefficients) for imino protons corroborated hydrogen bonding patterns observed in the NMR-derived structural model of the branch site helix. The ability to observe non-base-paired imino protons of RNA is of significant value in structure determination of RNA motifs containing loop and bulge regions.