Riboswitch Detection Using Profile Hidden Markov Models

Background  

Riboswitches are a type of noncoding RNA that regulate gene expression by switching from one structural conformation to another on ligand binding. The various classes of riboswitches discovered so far are differentiated by the ligand, which on binding induces a conformational switch. Every class of riboswitch is characterized by an aptamer domain, which provides the site for ligand binding, and an expression platform that undergoes conformational change on ligand binding. The sequence and structure of the aptamer domain is highly conserved in riboswitches belonging to the same class. We propose a method for fast and accurate identification of riboswitches using profile Hidden Markov Models (pHMM). Our method exploits the high degree of sequence conservation that characterizes the aptamer domain.     

A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.     

Hyperosmotic readjustment of the crab,Hemigrapsus edwardsi

Summary Daily changes in the composition of the haemolymph and muscle tissue ofHemigrapsus edwardsi, following transfer to hyperosmotic saline, were investigated. The haemolymph approached a new equilibrium, with respect to ionic constituents, within 48 h. The muscle fibre water content, expressed in kg/kg dry weight, fell initially and then rose. This was associated with a rise in the amount (per kg tissue dry weight) of intracellular ninhydrin-positive substances (mainly amino acids) which was substantially complete within four days. It was concluded that this crab is capable of relatively rapid intracellular readjustment to hyperosmotic stress.     

Conserved immunoglobulin-like features in a family of periplasmic pilus chaperones in bacteria.

Detailed structural analyses revealed a family of periplasmic chaperones in Gram-negative prokaryotes which are structurally and possibly evolutionarily related to the immunoglobulin superfamily and assist in the assembly of adhesive pili. The members of this family have similar structures consistent with the overall topology of an immunoglobulin fold. Seven pilus chaperone sequences from Escherichia coli, Haemophilus influenzae and Klebsiella pneumoniae were aligned and their consensus sequence was superimposed onto the known three-dimensional structure of PapD, a representative member of the family. The molecular details of the conserved and variable structural motifs in this family of periplasmic chaperones give important insight into their structure, function, mechanism of action and evolutionary relationship with the immunoglobulin superfamily.     

Adaptive Reversion of a Frameshift Mutation in Escherichia Coli

Mutation rates are generally thought not to be influenced by selective forces. This doctrine rests on the results of certain classical studies of the mutations that make bacteria resistant to phages and antibiotics. We have studied a strain of Escherichia coli which constitutively expresses a lacI-lacZ fusion containing a frameshift mutation that renders it Lac-. Reversion to Lac+ is a rare event during exponential growth but occurs in stationary cultures when lactose is the only source of energy. No revertants accumulate in the absence of lactose, or in the presence of lactose if there is another, unfulfilled requirement for growth. The mechanism for such mutation in stationary phase is not known, but it requires some function of RecA which is apparently not required for mutation during exponential growth.     

Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication.

Efficient expression of herpes simplex virus genes requires the synthesis of functional ICP4, a nuclear phosphoprotein that contains a prominent serine-rich region between amino acids 142 and 210. Residues in this region not only are potential sites for phosphorylation but also are involved in the functions of ICP4. By comparing the growth of a virus in which this region is deleted (d8-10) with wild-type virus (KOS) in PC12 cells or PC12 cells that are deficient in cyclic AMP-dependent protein kinase (PKA), two observations were made: (i) the growth of wild-type virus was impaired by 1 to 2 orders of magnitude in the PKA-deficient cells, indicating the involvement of PKA in the growth cycle of herpes simplex virus type 1, and (ii) while the growth of d8-10 was impaired by almost 2 orders of magnitude in wild-type cells, it was not further impaired (as was that of wild-type virus) in PKA-deficient cells, implicating the region deleted in d8-10 as a possible target for cellular PKA. In trigeminal'ganglia of mice, the d8-10 mutant virus grew poorly; however, it established latency in nearly 90% of ganglia tested. Studies of the phosphorylation of wild-type and d8-10 ICP4 proteins revealed that the serine-rich region is a major determinant for phosphorylation of ICP4 in vivo and that the phosphorylation state could change as a function of the PKA activity. Consistent with this observation, the serine-rich region of ICP4 was shown to be a target for PKA in vitro. While intact ICP4 was readily phosphorylated by ICP4 in vitro, the d8-10 mutant ICP4 was not. Moreover, a synthethic peptide representing a sequence in the serine tract that is predicted to be a substrate for PKA was phosphorylated by PKA in vitro, having a Km within the physiological range. These data suggest that PKA plays a role in viral growth through phosphorylation of one or more sites on the ICP4 molecule.     

Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.