A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein.

Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements. The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein. Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses. The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation. Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus. A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag. Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs. While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1. Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.     

Prokaryotic-style frameshifting in a plant translation system: conservation of an unusual single-tRNA slippage event

Ribosomal frameshifting signals are found in mobile genetic elements, viruses and cellular genes of prokaryotes and eukaryotes. Typically they comprise a slippery sequence, X XXY YYZ, where the frameshift occurs, and a stimulatory mRNA element. Here we studied the influence of host translational environment and the identity of slippery sequence-decoding tRNAs on the frameshift mechanism. By expressing candidate signals in Escherichia coli, and in wheatgerm extracts depleted of endogenous tRNAs and supplemented with prokaryotic or eukaryotic tRNA populations, we show that when decoding AAG in the ribosomal A-site, E.coli tRNA(Lys) promotes a highly unusual single-tRNA slippage event in both prokaryotic and eukaryotic ribosomes. This event does not appear to require slippage of the adjacent P-site tRNA, although its identity is influential. Conversely, asparaginyl-tRNA promoted a dual slippage event in either system. Thus, the tRNAs themselves are the main determinants in the selection of single- or dual-tRNA slippage mechanisms. We also show for the first time that prokaryotic tRNA(Asn) is not inherently 'unslippery' and induces efficient frameshifting when in the context of a eukaryotic translation system.     

Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus.

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.     

The yeast Ty element: recent advances in the study of a model retro-element

The past three years have seen a dramatic increase in our understanding of the structural organization and expression strategies of the dispersed, repetitive yeast transposon, Ty. These studies have led to a logical comparison of Ty with retroviral proviruses and other mobile, repetitive elements. Such comparisons have culminated in the hypotheses that transposition occurs via the formation of Ty-encoded virus-like particles and that these particles represent a basic unit of all ‘retro-systems’.     

Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5′ adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5′ and 3′ of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific ‘shifty’ codons.     

Isolation and characterization of RARE-1, a Ty1/copia-like retrotransposon domesticated in the genome of Rhizophora apiculata

Long terminal repeat (LTR) retrotransposons are predominant mobile elements that play important roles in plant genome evolution. Here, we isolated the first putative complete Ty1/copia-like retrotransposon of 6303 bp in mangrove Rhizophora apiculata, named RARE-1. RARE-1 was homologous to the soybean retroelement 1 (SORE-1) and exhibited abundant cis-regulatory motifs involved in various stress responses in its LTRs. Using the sequence-specific amplification polymorphism (S-SAP) technique, we obtained a total of 112 bands for two R. apiculata populations from Hainan, China and Ranong, Thailand. The Hainan population showed slightly higher S-SAP polymorphism but fewer unique bands than the Ranong population. Moreover, the Hainan population also had significantly more copies of RARE-1 than the Ranong population as revealed by quantitative real-time PCR (qPCR). Our results suggested that RARE-1 might have been domesticated in the R. apiculata genome, as a result of the long-term evolution of mangroves under the extreme environment.     

Mutation of a highly conserved base in the yeast mitochondrial 21S rRNA restricts ribosomal frameshifting

A mutation shown to cause resistance to chloramphenicol inSaccharomyces cerevisiae was mapped to the central loop in domain V of the yeast mitochondrial 21S rRNA. The mutant 21S rRNA has a base pair exchange from U₂₆₇₇ (corresponding to U₂₅₀₄ inEscherichia coli) to C₂₆₇₇, which significantly reduces rightward frameshifting at a UU UUU UCC A site in a + 1 U mutant. There is evidence to suggest that this reduction also applies to leftward frameshifting at the same site in a – 1 U mutant. The mutation did not increase the rate of misreading of a number of mitochondrial missense, nonsense or frameshift (of both signs) mutations, and did not adversely affect the synthesis of wild-type mitochondrial gene products. It is suggested here that ribosomes bearing either the C₂₆₇₇ mutation or its wild-type allele may behave identically during normal decoding and only differ at sites where a ribosomal stall, by permitting non-standard decoding, differentially affects the normal interaction of tRNAs with the chloramphenicol resistant domain V. Chloramphenicol-resistant mutations mapping at two other sites in domain V are described. These mutations had no effect on frameshifting.     

Structure of triphosphoryl nucleotide bound at the active site of yeast hexokinase: 1H-nuclear magnetic resonance study

Conformation of a nonhydrolyzable adenosine triphosphate (ATP) analogue, adenylyl-(,-methylene)-diphosphonate (AMPPCP) bound at the active site of yeast hexokinase-PII was determined by proton two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) and molecular dynamics simulations. The effect of the glucose-induced domain closure on the conformation of the nucleotide was evaluated by making measurements on two different complexes: PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II). TRNOE measurements were made at 500 MHz, 10°C, as a function of several mixing times varying in the range of 40 to 200 ms. Interproton distances derived from the analysis of NOE buildup curves were used as restraints in molecular dynamics simulations to determine the conformation of the enzyme bound nucleotide. The adenosine moiety was found to bind in high anti conformation with a glycosidic torsion angle = 48 ± 5 degrees in both complexes. However, significant differences in the conformations of the ribose and triphosphoryl chain of the nucleotide are observed between the two complexes. The phase angles of pseudorotation P in PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II) are 87 degrees and 77 degrees, describing a OE and OT₄ sugar pucker and the amplitudes of the sugar pucker () are 37 degrees and 61 degrees, respectively.