Recognition of nucleoside triphosphates during RNA-catalyzed primer extension.

In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K(m) and a higher k(cat), suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the gamma-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k(cat) of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.

Follicular stromal cells and lymphocyte homing to follicles.

Follicular dendritic cells (FDCs), the best defined stromal cell subset within lymphoid follicles, play a critical role in presenting intact antigen to B lymphocytes. The discovery that many follicular stromal cells make B-lymphocyte chemoattractant (BLC), a CXC chemokine that attracts CXCR5+ cells, provides a basis for understanding how motile B cells come into contact with stationary FDCs. Here we review our work on BLC and discuss properties of BLC-expressing follicular stromal cells. We also review the properties of primary follicle and germinal center FDCs and suggest a model of FDC development that incorporates information about BLC expression. Finally, we consider how antigen recognition causes T and B lymphocytes to undergo changes in chemokine responsiveness that may help direct their movements into, or out of, lymphoid follicles.

A B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1.

Secondary lymphoid organs (spleen, lymph nodes and Peyer's patches) are divided into compartments, such as B-cell zones (follicles) and T-cell zones, which provide specialized environments for specific steps of the immune response. Migration of lymphocyte subsets into these compartments is essential for normal immune function, yet the molecular cues guiding this cellular traffic are poorly defined. Chemokines constitute a family of chemotactic cytokines that have been shown to direct the migration of leukocytes during inflammation and which may be involved in the constitutive homing of lymphocytes into follicles and T-cell zones. Here we describe a novel chemokine, B-lymphocyte chemoattractant (BLC), that is strongly expressed in the follicles of Peyer's patches, the spleen and lymph nodes. BLC strongly attracts B lymphocytes while promoting migration of only small numbers of T cells and macrophages, and therefore is the first chemokine to be identified that is selective towards B cells. An orphan chemokine receptor, Burkitt's lymphoma receptor 1 (BLR-1), has been found to be required for B-cell migration into lymphoid follicles. We show that BLC stimulates calcium influx into, and chemotaxis of, cells transfected with BLR-1. Our results indicate that BLC functions as a BLR-1 ligand and may guide B lymphocytes to follicles in secondary lymphoid organs.

RNA-catalysed RNA polymerization using nucleoside triphosphates.

The hypothesis that certain RNA molecules may be able to catalyse RNA replication is central to current theories of the early evolution of life. In support of this idea, we describe here an RNA that synthesizes RNA using the same reaction as that employed by protein enzymes that catalyse RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of up to six mononucleotides. The added nucleotides are joined to the growing RNA chain by 3',5'-phosphodiester linkages. The ribozyme shows marked template fidelity: extension by nucleotides complementary to the template is up to 1,000 times more efficient than is extension by mismatched nucleotides.

The secondary structure and sequence optimization of an RNA ligase ribozyme.

In vitro selection can generate functional sequence variants of an RNA structural motif that are useful for comparative analysis. The technique is particularly valuable in cases where natural variation is unavailable or non-existent. We report the extension of this approach to a new extreme--the identification of a 112 nt ribozyme secondary structure imbedded within a 186 nt RNA. A pool of 10(14) variants of an RNA ligase ribozyme was generated using combinatorial chemical synthesis coupled with combinatorial enzymatic ligation such that 172 of the 186 relevant positions were partially mutagenized. Active variants of this pool were enriched using an in vitro selection scheme that retains the sequence variability at positions very close to the ligation junction. Ligases isolated after four rounds of selection catalyzed self-ligation up to 700 times faster than the starting sequence. Comparative analysis of the isolates indicated that when complexed with substrate RNAs the ligase forms a nested, double pseudo-knot secondary structure with seven stems and several important joining segments. Comparative analysis also suggested the identity of mutations that account for the increased activity of the selected ligase variants; designed constructs incorporating combinations of these changes were more active than any of the individual ligase isolates.

Structurally complex and highly active RNA ligases derived from random RNA sequences.

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.