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PubMed2004

Classification of common functional loops of kinase super-families.

A structural classification of loops has been obtained from a set of 141 protein structures classified as kinases. A total of 1813 loops was classified into 133 subclasses (9 betabeta(links), 15 betabeta(hairpins), 31 alpha-alpha, 46 alpha-beta and 32 beta-alpha). Functional information and specific features relating subclasses and function were included in the classification. Functional loops such as the P-loop (shared by different folds) or the Gly-rich-loop, among others, were classified into structural motifs. As a result, a common mechanism of catalysis and substrate binding was proved for most kinases. Additionally, the multiple-alignment of loop sequences made within each subclass was shown to be useful for comparative modeling of kinase loops. The classification is summarized in a kinase loop database located at http://sbi.imim.es/archki.

Amino Acid Motifs↗
PubMed2005

Crystal packing effects on protein loops.

The effects of crystal packing on protein loop structures are examined by (1) a comparison of loops in proteins that have been crystallized in alternate packing arrangements, and (2) theoretical prediction of loops both with and without the inclusion of the crystal environment. Results show that in a minority of cases, loop geometries are dependent on crystal packing effects. Explicit representation of the crystal environment in a loop prediction algorithm can be used to model these effects and to reconstruct the structures, and relative energies, of a loop in alternative packing environments. By comparing prediction results with and without the inclusion of the crystal environment, the loop prediction algorithm can further be used to identify cases in which a crystal structure does not represent the most stable state of a loop in solution. We anticipate that this capability has implications for structural biology.

Algorithms↗
PubMed1991

The crystal structure of the "open" and the "closed" conformation of the flexible loop of trypanosomal triosephosphate isomerase.

Triosephosphate isomerase has an important loop near the active site which can exist in a "closed" and in an "open" conformation. Here we describe the structural properties of this "flexible" loop observed in two different structures of trypanosomal triosephosphate isomerase. Trypanosomal triosephosphate isomerase, crystallized in the presence of 2.4 M ammonium sulfate, packs as an asymmetric dimer of 54,000 Da in the crystallographic asymmetric unit. Due to different crystal contacts, peptide 167-180 (the flexible loop of subunit-1) is an open conformation, whereas in subunit-2, this peptide (residues 467-480) is in a closed conformation. In the closed conformation, a hydrogen bond exists between the tip of the loop and a well-defined sulfate ion which is bound to the active site of subunit-2. Such an active site sulfate is not present in subunit-1 due to crystal contacts. When the native (2.4 M ammonium sulfate) crystals are transferred to a sulfate-free mother liquor, the flexible loop of subunit-2 adopts the open conformation. From a closed starting model, this open conformation was discovered through molecular dynamics refinement without manual intervention, despite involving C alpha shifts of up to 7 A. The tip of the loop, residues 472, 473, 474, and 475, moves as a rigid body. Our analysis shows that in this crystal form the flexible loop of subunit-2 faces a solvent channel. Therefore the open and the closed conformations of this flexible loop are virtually unaffected by crystal contacts. The actual observed conformation depends only on the absence or presence of a suitable ligand in the active site.

Amino Acid Sequence↗
PubMed2000

Structure-function relationships of the two surface loops of myosin heavy chain isoforms from thermally acclimated carp.

The structure-function relationships of fast skeletal myosin isoforms remain poorly understood. To shed some light, we constructed chimeric myosins comprised of Dictyostelium myosin heavy chain backbone with carp loop sequences and analyzed their functional properties. A loop 2-10 chimeric myosin having the loop 2 sequence of the fast skeletal isoform predominantly expressed in carp acclimated to 10 degrees C showed V(max) in actin-activated Mg(2+)-ATPase activity 1.4-fold higher than a loop 2-30 chimera constructed from the loop 2 sequence of the dominant isoform in carp acclimated to 30 degrees C. These two chimera exhibited no significant differences in sliding velocity of actin filaments in in vitro motility assay. Contrastingly, both loop 1-associated chimeras, loop 1-10 and loop 1-30, did not differ in both ATPase activity and in sliding velocity of actin filaments.

Acclimatization↗
PubMed1996

Left-right asymmetric localization of flectin in the extracellular matrix during heart looping.

The early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left-right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere, in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewska et al., 1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8- (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left-right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twal et al., 1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly.

Animals↗
PubMed1997

Management of cervical intraepithelial neoplasia during pregnancy with LOOP excision.

OBJECTIVE: Management of severe cervical dysplasia/possible microinvasive carcinoma during pregnancy is frequently associated with significant morbidity. The purpose of this study is to determine the efficacy of LOOP excision performed during pregnancy, and also to record the nature and frequency of complications of the procedure. METHODS: Twenty women underwent LOOP excision during pregnancy. The gestational age range was 8-34 weeks. Data concerning indications, complications, and histopathologic results were recorded. RESULTS: Fourteen of 20 (70%) had dysplastic changes in the LOOP specimen. Eight of 14 (57%) had involved margins. Nine of 19 (47%) had residual dysplasia 3 months postpartum, including 3 patients whose initial LOOP specimens were negative for dysplasia. Significant morbidity included 3 preterm births, 2 patients who required blood transfusion following LOOP, and 1 unexplained intrauterine fetal demise documented 4 weeks post-LOOP. The gestational age range of those patients who had significant morbidity was 27-34 weeks. CONCLUSIONS: LOOP excision of the cervix during pregnancy does not consistently produce diagnostic specimens and is associated with a significant rate of residual disease. Morbidity appears similar to that of cone biopsy during pregnancy and occurs primarily when the procedure is performed in the third trimester. Until refinements in technique occur, LOOP excision during pregnancy should be reserved for limited indications.

Adult↗
PubMed1999

A study of diagnostic failure of loop conization in microinvasive carcinoma of the cervix.

OBJECTIVE: The aim of this study was to evaluate the specimen adequacy and the histological interpretation of loop conization for microinvasive cervical carcinoma. METHODS: We retrospectively reviewed the histopathological findings of the original cone specimens together with the final hysterectomy specimens in patients with microinvasive carcinoma of the cervix. From 1990 to 1995, 63 consecutive patients with microinvasive carcinoma of the cervix were included in the study, of which 35 patients underwent loop conization and 28 underwent cold-knife conization. All patients had a hysterectomy. RESULTS: The mean width, depth, and cone volume of the conization specimens were 2.44 cm, 2.15 cm, and 3.96 cm3, respectively, in the loop group versus 2.3 cm, 2.35 cm, and 4.38 cm3 in the cold-knife group. No significant differences were seen between the two groups. The application of loop conization was completed in a single slice in 27 patients (77.1%) and multiple slices by the loop in 8 (22.9%), in spite of the attempt to perform conization in a one-pass application when possible. In assessing these cone specimens microscopically, the rate of transection of tissue was significantly higher in the loop cone than in the cold-knife cone (17.1% versus 0%, P = 0.02). Because of transection of tissue and misorientation, pathologic determination of the depth and width of stromal invasion was undetermined in two loop cone specimens compared with none in the cold-knife cones. CONCLUSION: Our study suggests that cold-knife conization is a preferred method in assessing microinvasive carcinoma of the cervix if multiple applications of loop conization are inevitable.

Adult↗
PubMed1993

Erythromycin binding is reduced in ribosomes with conformational alterations in the 23 S rRNA peptidyl transferase loop.

The antibiotic erythromycin inhibits protein synthesis by binding to the 50 S ribosomal subunit, where the drug interacts with the unpaired bases 2058A and 2059A in the peptidyl transferase loop of 23 S rRNA. We used a chemical modification approach to analyse conformational changes that are induced by mutations in the peptidyl transferase loop, and to determine how these changes affect drug interaction. Mutations at positions 2057 (G-->A) and 2058 (A-->G, or -->U), all of which confer drug resistance, induce a more open conformation in the peptidyl transferase loop. Erythromycin still protects against chemical modification in the mutant peptidyl transferase loops, but the affinity of the drug interaction is reduced 20-fold in the 2057A mutant, 10(3)-fold in the 2058U mutant and 10(4)-fold in the 2058G mutant. Single mutations at position 2032 in the adjacent hairpin loop, which have previously been shown to alter drug tolerances, gave no detectable effects on the structure of the peptidyl transferase loop or on erythromycin binding. Dual mutations at positions 2032 and 2058, however, induce a marked change in the rRNA conformation with opening of the phylogenetically conserved base-pair 2063C.2447G, and confer a slow growth, drug-sensitive phenotype. The data suggest that the target site of erythromycin lies within the peptidyl transferase loop, and that limited disruption of the conformation of this site reduces drug binding, and consequently confers resistance. In addition, there is structurally and functionally important interaction between the drug target site in the peptidyl transferase loop and position 2032.

Base Sequence↗
PubMed1996

The influence of symmetric internal loops on the flexibility of RNA.

Internal loops are structural elements, often highly conserved, that are found in many RNA molecules of biological importance. They consist of short stretches of sequence in which the bases in one strand are not able to form canonical pairs with bases in the other strand, and are bounded on either side by helical RNA. In an effort to examine the influence of internal loops on the relative angular orientations of the flanking helices, we have quantified the apparent bend angles for symmetric internal loops of the form A(n)-A(n) and U(n)-U(n) (n=2, 4, and 6), located at the center of 150 to 154 bp RNA molecules, using the method of transient electric birefringence. This hydrodynamic method exploits the extreme sensitivity of the rate of rotational reorientation of the RNA molecules to the presence and magnitude of internal bends and/or points of increased flexibility. The birefringence decay behavior of the loop-containing RNA molecules was found to be much less strongly influenced by the presence of symmetric internal loops than by bulges of the same sequence and size. This general observation is mirrored by the electrophoretic behavior of the loop-containing molecules, which are much less strongly retarded on polyacrylamide gels than are corresponding, bulge-containing RNA molecules. The apparent bend angles for the symmetric loops range from approximately 20 degrees to 40 degrees as n is increased 2 to 6 with a marginal shift to smaller angles in the presence of Mg2+. The apparent angles were similar when represented either as fixed bends of the specified angles (static representation), or as points of increased flexibility of specified root-mean-square angle (dynamic representation). For the. For the latter representation, the corresponding angular dispersion would correspond to a loop persistence length of approximately 60 to 150 A, compared to 700 A for duplex RNA and depending slightly on sequence and buffer conditions.

Base Sequence↗
PubMed1997

An automated classification of the structure of protein loops.

Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a non-redundant set of 233 proteins with less than 25% sequence homology (X-ray resolution better than 2.5 A). Loops have been classified into five types (alpha-alpha, beta-beta links, beta-beta hairpins, alpha-beta and beta-alpha) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (phi, psi) are used to define the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 alpha-alpha, 11 beta-beta links, 14 beta-beta hairpins, 13 alpha-beta and 9 beta-alpha) were identified with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and finds other clusters that have hitherto not been described. Consensus sequence patterns were identified for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.

Crystallography, X-Ray↗
PubMed1997

Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs.

Almost all transfer RNA molecules sequenced so far contain two universal modified nucleosides at positions 54 and 55, respectively: ribothymidine (T54) and pseudouridine (psi 55). To identify the tRNA elements recognized by tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase from the yeast Saccharomyces cerevisiae, a set of 43 yeast tRNA(Asp) mutants were used. Some variants contained point mutations, while the others included progressive reductions in size down to a tRNA minisubstrate consisting of the T psi-loop with only one G.C base-pair as stem (9-mer). All substrates (full-sized tRNA(Asp) and various minihelices) were produced in vitro by T7 transcription and tested using yeast extract (S100) as a source of enzymatic activities and S-adenosyl-L-methionine as a methyl donor. The results indicate that the minimal substrate for enzymatic formation of psi 55 is a stem/loop structure with only four G.C base-pairs in the stem, while a longer stem is required for efficient T54 formation. None of the conserved nucleotides (G53, C56, A58 and C61) and U54 for psi 55 or U55 for T54 formation can be replaced by any of the other three canonical nucleotides. Yeast tRNA:m5uridine-54 methyltransferase additionally requires the presence of a pyrimidine-60 in the loop. Interestingly, in a tRNA(Asp) variant in which the T psi-loop was permuted with the anticodon-loop, the new U32 and U33 residues derived from the T psi-loop were quantitatively converted to T32 and psi 33, respectively. Structural mapping of this variant with ethylnitrosourea confirmed that the intrinsic characteristic structure of the T psi-loop was conserved upon permutation and that the displaced anticodon-loop did not acquire a T psi-loop structure. These results demonstrate that a local conformation rather than the exact location of the U-U sequence within the tRNA architecture is the important identity determinant for recognition by yeast tRNA:m5uridine-54 methyltransferase and tRNA:pseudouridine-55 synthase.

Base Sequence↗
PubMed1999

Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA.

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.

Base Sequence↗
PubMed1999

The importance of internal loops within RNA substrates of ADAR1.

Adenosine deaminases that act on RNA (ADARs) are a family of RNA editing enzymes that convert adenosines to inosines within double-stranded RNA (dsRNA). Although ADARs deaminate perfectly base-paired dsRNA promiscuously, deamination is limited to a few, selected adenosines within dsRNA containing mismatches, bulges and internal loops. As a first step in understanding how RNA structural features promote selectivity, we investigated the role of internal loops within ADAR substrates. We observed that a dsRNA helix is deaminated at the same sites whether it exists as a free molecule or is flanked by internal loops. Thus, internal loops delineate helix ends for ADAR1. Since ADAR1 deaminates short RNAs at fewer adenosines than long RNAs, loops decrease the number of deaminations within an RNA by dividing a long RNA into shorter substrates. For a series of symmetric internal loops related in sequence, larger loops (>/=six nucleotides) acted as helix ends, whereas smaller loops (</=four nucleotides) did not. Our work provides the first information about how secondary structure within ADAR substrates dictates selectivity, and suggests a rational approach for delineating minimal substrates for RNAs deaminated by ADARs in vivo.

Adenosine Deaminase↗
PubMed1999

A DNA hairpin with a single residue loop closed by a strongly distorted Watson-Crick G x C base-pair.

Our previous NMR and modeling studies have shown that the single-stranded 19mer oligonucleotides d(AGCTTATC-ATC-GATAA GCT) -ATC- and d(AGCTTATC-GAT-GATAAGCT) -GAT- encompassing the strongest topoisomerase II cleavage site in pBR322 DNA could form stable hairpin structures. A new sheared base-pair, the pyrimidine-purine C x A, was found to close the single base -ATC- loop, while -GAT- displayed a flexible loop of three/five residues with no stabilizing interactions. Now we report a structural study on -GAC-, an analog of -GAT-, derived through the substitution of the loop residue T by C. The results obtained from NMR, non-denaturing PAGE, UV-melting, circular dichroism experiments and restrained molecular dynamics indicate that -GAC- adopts a hairpin structure folded through a single residue loop. In the -GAC- hairpin the direction of the G9 sugar is reversed relative to the C8 sugar, thus pushing the backbone of the loop into the major groove. The G9 x C11 base-pair closing the loop is thus neither a sheared base-pair nor a regular Watson-Crick one. Although G9 and C11 are paired through hydrogen bonds of Watson-Crick type, the base-pair is not planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. The distortion decreases the sugar C1'-C1' distance between the paired G9 and C11, to 8 A versus 11 A in the standard B-DNA. The A10 residue at the center of the loop interacts with the G9 x C11 base-pair, and seems to contribute to the extra thermal stability displayed by -GAC- compared to -GAT-. Test calculations allowed us to identify the experimental NOEs critical for inducing the distorted G.C Watson-Crick base-pair. The preference of -GAC- for a hairpin structure rather than a duplex is confirmed by the diffusion constant values obtained from pulse-field gradient NMR experiments. All together, the results illustrate the high degree of plasticity of single-stranded DNAs which can accommodate a variety of turn-loops to fold up on themselves.

Base Pairing↗
PubMed2000

High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence.

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and structural consequences of truncating the binding loop residues to alanine. In addition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P(1) and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 30 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collected to 1.4 A at room temperature allowed anisotropic structure refinement characterized by R=0.1048. The structure reveals all 58 residues, including the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct chiralities. This bridge, together with an internal water molecule, contributes to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C(alpha) atoms of 0.7 A at Ala13). Four (instead of the typical three) additional water molecules are buried in an internal cleft and connected to the surface via a sulfate anion. Three more SO(4)(2-) anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimeric structure between symmetry-related BPTI molecules, in which electrostatic and hydrogen bonding interactions resulting from the mutated Lys15Arg substitution are of central importance. This dimeric interaction involves direct recognition loop-recognition loop contacts, part of which are hydrophobic interactions of the patches created by the alanine mutations. Another 2-fold symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjacent intramolecular beta-hairpin loops, creates a four-stranded structure.

Alanine↗
PubMed2002

Conformational change of the E-F interhelical loop in the M photointermediate of bacteriorhodopsin.

The conformation of the structured EF interhelical loop of bacteriorhodopsin and its change in the M photointermediate were assessed by measuring the rate of reaction of 16 single engineered cysteine residues along the loop with water-soluble sulfhydryl reagents. The exposure to the bulk in the unilluminated state determined with the cysteine reaction correlated well with the degree of access to water calculated from the crystallographic structure of the loop. The EF-loop should be affected by the well-known outward tilt of helix F in the M and N intermediates of the photocycle. A second mutation in each cysteine mutant, the D96N residue replacement, allowed full conversion to the M state by illumination. The reaction rates measured under these conditions indicated that buried residues tend to become more exposed, and exposed residues become more buried in M. This is to be expected from tilt of helix F. However, the observation of increased exposure of four residues near the middle of the loop, where steric effects are only from other loop residues, indicate that the conformation of the EF-loop itself is changed. Thus, the motion of the loop in M is more complex than expected from simple tilt of helix F, and may include rotation that unwinds its twist.

Bacteriorhodopsins↗
PubMed2001

Molecular systematics of the western rattlesnake, Crotalus viridis (Viperidae), with comments on the utility of the D-loop in phylogenetic studies of snakes.

Crotalus viridis, the western rattlesnake, ranges throughout western North America and has been divided into at least eight subspecies. However, the validity of and relationships among these subspecies and the monophyly of C. viridis as a whole are questionable. We used mitochondrial DNA sequence data from the D-loop region and ND2 gene to examine the relationships among 26 populations of C. viridis and to test the monophyly of this species. These data were analyzed separately and combined using maximum-likelihood and maximum-parsimony. The C. viridis group was monophyletic in all combined analyses, consisting of two strongly divergent clades. We recommend that these clades be recognized as two distinct evolutionary species: C. viridis and C. oreganus. Crotalus viridis should be restricted to the subspecies viridis and nuntius and the remaining subspecies be assigned to the species C. oreganus. Our data do not allow strong evaluation of the validity of the subspecies. We found that the ND2 gene had greater sequence divergences among closely related individuals than the D-loop region, but this relationship reversed at higher levels of divergence. This pattern is apparently due to: (1) ND2 third positions evolving faster than the D-loop but becoming saturated at higher levels of divergence, and (2) the D-loop evolving faster than ND2 second (and possibly first) positions. Our results suggest that the ND2 gene is preferable for examining intraspecific relationships and the D-loop may better resolve relationships between species of snakes. The latter result is contrary to the common perception of the phylogenetic utility of the D-loop. Another unusual result is that the 145 bp spacer region, adjacent to the 5' end of the light strand of the D-loop, provides greater phylogenetic resolution than the 1030 bp D-loop.

Animals↗
PubMed1995

Reciprocal relationship between stem-loop potential and substitution density in retroviral quasispecies under positive Darwinian selection.

Nucleic acids have the potential to form intrastrand stem-loops if complementary bases are suitably located. Computer analyses of poliovirus and retroviral RNAs have revealed a reciprocal relationship between "statistically significant" stem-loop potential and "sequence variability." The statistically significant stem-loop potential of a nucleic acid segment has been defined as a function of the difference between the folding energy of the natural segment (FONS) and the mean folding energy of a set of randomized (shuffled) versions of the natural segment (FORS-M). Since FONS is dependent on both base composition and base order, whereas FORS-M is solely dependent on base composition (a genomic characteristic), it follows that statistically significant stem-loop potential (FORS-D) is a function of base order (a local characteristic). In retroviral genomes, as in all DNA genomes studied, positive FORS-D values are widely distributed. Thus there have been pressures on base order both to encode specific functions and to encode stem-loops. As in the case of DNA genomes under positive Darwinian selection pressure, in HIV-1 specific function appears to dominate in rapidly evolving regions. Here high sequence variability, expressed as substitution density (not indel density), is associated with negative FORS-D values (impaired base-order-dependent stem-loop potential). This suggests that in these regions HIV-1 genomes are under positive selection pressure by host defenses. The general function of stem-loops is recombination. This is a vital process if, from among members of viral "quasispecies," functional genomes are to be salvaged. Thus, for rapidly evolving RNA genomes, it is as important to conserve base-order-dependent stem-loop potential as to conserve other functions.

Base Sequence↗