ABSTRACT
The capsaicin receptor (VR1, TRPV1) is a ligand-gated ion channel predominantly expressed in peripheral nociceptors and activated by multiple noxious stimuli including products of inflammation. A 5'-splice variant (VR.5'sv) of TRPV1 has been previously isolated and found to be insensitive to noxious stimuli. We report in this study that coexpression of VR.5'sv with TRPV1 in Xenopus oocytes blocks TRPV1-mediated current responses. Oocytes expressing the inhibitory profile demonstrated colocalization of TRPV1 and VR.5'sv-associated immunostaining in the plasma membrane. TRPV1 protein expression was comparable in all groups. Evidence of endogenous VR.5'-splice variant-like-protein expression was detected in dorsal root ganglion. These results support the idea that coexpression of VR.5'sv or a similar variant could result in inhibitory modulation of TRPV1 activation.
Subject(s)
Protein Isoforms/metabolism , TRPV Cation Channels/metabolism , Analysis of Variance , Animals , Biotinylation/methods , Ganglia, Spinal/cytology , Gene Expression , Hot Temperature , Membrane Potentials/drug effects , Membrane Potentials/physiology , Microinjections , Neurons/drug effects , Neurons/physiology , Oocytes , Patch-Clamp Techniques , Rats , XenopusABSTRACT
The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).
Subject(s)
Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Escherichia coli/chemistry , Protein Biosynthesis , RNA, Bacterial/chemistry , RNA, Messenger/chemistry , Aminoglycosides/chemistry , Aminoglycosides/metabolism , Anti-Bacterial Agents/chemistry , Base Sequence , Binding Sites , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Ribosomes/genetics , Ribosomes/metabolism , Structure-Activity Relationship , Templates, GeneticABSTRACT
We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.
Subject(s)
Escherichia coli/chemistry , Escherichia coli/ultrastructure , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Ribosomes/ultrastructure , Binding Sites , Crystallization , Crystallography, X-Ray , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/chemistry , Hydrogen Bonding , Magnesium/metabolism , Models, Molecular , Nucleic Acid Conformation , Peptidyl Transferases/chemistry , Protein Biosynthesis , Protein Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolismABSTRACT
During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures. Furthermore, protein Y competes with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature.
