scholarly journals Inhibition of Translation Termination Mediated by an Interaction of Eukaryotic Release Factor 1 with a Nascent Peptidyl-tRNA

2002 ◽  
Vol 22 (24) ◽  
pp. 8562-8570 ◽  
Author(s):  
Deanna M. Janzen ◽  
Lyudmila Frolova ◽  
Adam P. Geballe
Keyword(s):  
Translation Termination ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Dependent Manner ◽  
Release Factor ◽  
Inhibitory Mechanism ◽  
Reading Frame ◽  
Nascent Peptide ◽  
Peptide Product

ABSTRACT Expression of the human cytomegalovirus UL4 gene is inhibited by translation of a 22-codon-upstream open reading frame (uORF2). The peptide product of uORF2 acts in a sequence-dependent manner to inhibit its own translation termination, resulting in persistence of the uORF2 peptidyl-tRNA linkage. Consequently, ribosomes stall at the uORF2 termination codon and obstruct downstream translation. Since termination appears to be the critical step affected by translation of uORF2, we examined the role of eukaryotic release factors 1 and 3 (eRF1 and eRF3) in the inhibitory mechanism. In support of the hypothesis that an interaction between eRF1 and uORF2 contributes to uORF2 inhibitory activity, specific residues in each protein, glycines 183 and 184 of the eRF1 GGQ motif and prolines 21 and 22 of the uORF2 peptide, were found to be necessary for full inhibition of downstream translation. Immunoblot analyses revealed that eRF1, but not eRF3, accumulated in the uORF2-stalled ribosome complex. Finally, increased puromycin sensitivity was observed after depletion of eRF1 from the stalled ribosome complex, consistent with inhibition of peptidyl-tRNA hydrolysis resulting from an eRF1-uORF2 peptidyl-tRNA interaction. These results reveal the paradoxical potential for interactions between a nascent peptide and eRF1 to obstruct the translation termination cascade.

scholarly journals Abundant Early Expression of gpUL4 from a Human Cytomegalovirus Mutant Lacking a Repressive Upstream Open Reading Frame

2001 ◽  
Vol 75 (15) ◽  
pp. 7188-7192 ◽  
Author(s):  
John P. Alderete ◽  
Stephanie J. Child ◽  
Adam P. Geballe
Keyword(s):  
Cell Culture ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Human Fibroblasts ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Inhibitory Mechanism ◽  
Reading Frame ◽  
Early Expression ◽  
Translational Level

ABSTRACT The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.

scholarly journals A Premature Termination Codon Interferes with the Nuclear Function of an Exon Splicing Enhancer in an Open Reading Frame-Dependent Manner

1999 ◽  
Vol 19 (3) ◽  
pp. 1640-1650 ◽  
Author(s):  
Anand Gersappe ◽  
David J. Pintel
Keyword(s):  
Translation Termination ◽  
Rna Stability ◽  
Open Reading Frame ◽  
Termination Codon ◽  
Dependent Manner ◽  
Enhancer Element ◽  
Reading Frame ◽  
Exon Splicing ◽  
Nuclear Rna ◽  
Splicing Enhancer

ABSTRACT Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5′ element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5′ and 3′ elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3′ enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3′ splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

scholarly journals Translational Effects of Mutations and Polymorphisms in a Repressive Upstream Open Reading Frame of the Human Cytomegalovirus UL4 Gene

1999 ◽  
Vol 73 (10) ◽  
pp. 8330-8337 ◽  
Author(s):  
John P. Alderete ◽  
Sohail Jarrahian ◽  
Adam P. Geballe
Keyword(s):  
Human Cytomegalovirus ◽  
Stop Codon ◽  
Translation Termination ◽  
Laboratory Strain ◽  
Open Reading Frame ◽  
Peptide Sequence ◽  
Upstream Open Reading Frame ◽  
Missense Mutations ◽  
Reading Frame ◽  
Amino Terminal

ABSTRACT The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, ∼15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.

scholarly journals Sucrose Control of Translation Mediated by an Upstream Open Reading Frame-Encoded Peptide

2009 ◽  
Vol 150 (3) ◽  
pp. 1356-1367 ◽  
Author(s):  
Fatemeh Rahmani ◽  
Maureen Hummel ◽  
Jolanda Schuurmans ◽  
Anika Wiese-Klinkenberg ◽  
Sjef Smeekens ◽  
...  
Keyword(s):  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Reading Frame

scholarly journals Translational regulation of Arabidopsis XIPOTL1 is modulated by phosphocholine levels via the phylogenetically conserved upstream open reading frame 30

2012 ◽  
Vol 63 (14) ◽  
pp. 5203-5221 ◽  
Author(s):  
Fulgencio Alatorre-Cobos ◽  
Alfredo Cruz-Ramírez ◽  
Celine A. Hayden ◽  
Claudia-Anahí Pérez-Torres ◽  
Anne-Laure Chauvin ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Open Reading Frame ◽  
Upstream Open Reading Frame ◽  
Reading Frame
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