The Experts below are selected from a list of 138 Experts worldwide ranked by ideXlab platform
Roger W. Hendrix - One of the best experts on this subject based on the ideXlab platform.
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a balanced ratio of proteins from gene g and Frameshift extended gene gt is required for phage lambda tail assembly
Journal of Molecular Biology, 2013Co-Authors: Roger W. Hendrix, Robert L DudaAbstract:In bacteriophage λ, the overlapping open reading frames G and T are expressed by a programmed Translational Frameshift similar to that of the gag-pol genes of many retroviruses to produce the proteins gpG and gpGT. An analogous Frameshift is widely conserved among other dsDNA tailed phages in their corresponding "G" and "GT" tail genes even in the absence of detectable sequence homology. The longer protein gpGT is known to be essential for tail assembly, but the requirement for the shorter gpG remained unclear because mutations in gene G affect both proteins. A plasmid system that can direct the efficient synthesis of tails was created and used to show that gpG and gpGT are both essential for correct tail assembly. Phage complementation assays under conditions where levels of plasmid-expressed gpG or gpGT could be altered independently revealed that the correct molar ratio of these two related proteins, normally determined by the efficiency of the Frameshift, is also crucial for efficient assembly of functional tails. Finally, the physical connection between the G and T domains of gpGT, a consequence of the Frameshift mechanism of protein expression, appears to be important for efficient tail assembly.
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conserved Translational Frameshift in dsdna bacteriophage tail assembly genes
Molecular Cell, 2004Co-Authors: Roger W. Hendrix, Robert L DudaAbstract:Abstract A programmed Translational Frameshift similar to Frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage λ, this Frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous −1 Frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous −2 Frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this Frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.
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A programmed Translational Frameshift is required for the synthesis of a bacteriophage λ tail assembly protein
Journal of Molecular Biology, 1993Co-Authors: Margaret E. Levin, Roger W. Hendrix, Sherwood R CasjensAbstract:Abstract Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage λ tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a Translational Frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The Frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys in both the zero and -1 reading frames. The Frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for Frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance Frameshifting, as is seen in other well-characterized Frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the Frameshifting rate. Our results together with those of Katsura and Kuhl show that the Frameshift product, gpG-T, has an essential role in λ tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.
Emanuel Goldman - One of the best experts on this subject based on the ideXlab platform.
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evidence that uncharged trna can inhibit a programmed Translational Frameshift inescherichia coli
Journal of Molecular Biology, 1995Co-Authors: Wenwu Gao, Hieronim Jakubowski, Emanuel GoldmanAbstract:In the modified release factor 2 (RF2) programmed Translational Frameshift (with a sense codon replacing the wild-type in-frame UGA codon at the shift site), ribosomes shift +1 into the reading frame for an out-of-frame reporter fused to the Frameshift sequence. Partitioning of ribosomes between the out-of-frame shift and in-frame reading depends on the codon at the shift site and on the levels of tRNA decoding the in-frame codon. Overexpression of a tRNA species cognate to the in-frame codon at the shift site significantly reduces the frequency of frame-shifting, presumably by facilitating in-frame reading, which would reduce production of the out-of-frame reporter. However, since overexpression of a tRNA increases levels of both charged and uncharged tRNA, it is possible that uncharged cognate tRNA might be able to reduce the frequency of the Frameshift, by entering the A site on the ribosome. To test this, we manipulated charged and uncharged tRNA levels in vivo, using the tryptophan analog tryptophan hydroxamate, which increases the proportion of uncharged tRNA(Trp) by competing with cognate amino acid tryptophan for tryptophanyl-tRNA synthetase, thereby reducing protein synthesis. We report here that a slight but reproducible reduction in the relative frequency of the Frameshift is observed when tryptophan hydroxamate is added to cells containing the modified RF2 shift with UGG (Trp codon) at the shift site. When tRNA(Trp) is overexpressed from another plasmid, the shift frequency drops three- to fourfold, as expected, however, this reduction is still seen in the presence of the analog. Thus, under conditions when most of the tRNA(Trp) is apparently uncharged, excess tRNA(Trp) still causes a significant reduction in the Frameshift when UGG is at the shift site, providing evidence that uncharged cognate tRNA also can inhibit this Frameshift.
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increased ribosomal accuracy increases a programmed Translational Frameshift in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: J Sipley, Emanuel GoldmanAbstract:Abstract We have tested the effect of increased ribosomal fidelity on a modified version of the programmed release factor 2 (RF2) Translational Frameshift. In the constructs tested, the original UGA codon at the site of the shift was replaced by either of two sense codons, UGG (tryptophan), which allows a Frameshift of approximately 13%, or CUG (leucine), which allows a Frameshift of only approximately 2%. We confirmed the results of Curran and Yarus [Curran, J. F. & Yarus, M. (1989) J. Mol. Biol. 209, 65-77] in a wild-type ribosomal host, including a reduction of the UGG shift following induction of tRNA(Trp) from a plasmid copy of the tRNA gene. But to our surprise, in a hyperaccurate streptomycin pseudo-dependent host, the UGG Frameshift increased to more than 50%. When we added a tRNA(Trp) plasmid to these cells, induction of the tRNA(Trp) gene reduced the shift back to approximately 7%. Messenger RNA levels did not vary greatly under these different induced conditions. Other increased accuracy alleles also showed increased Frameshifting with UGG at the Frameshift site. All increased accuracy alleles led to slower translation rates, and there appeared to be a proportionality between the extent of reduction of synthesis for the in-frame reporter and the extent of UGG Frameshift for the out-of-frame reporter. There were little effects of increased accuracy on the lower level CUG Frameshift. However, over-production of the cognate tRNA(1Leu) dramatically reduced even this lower level of shift, despite the fact that tRNA(1Leu) is already the most abundant isoacceptor in Escherichia coli. These results can be rationalized by following the hypothesis of Curran and Yarus as follows: with wild-type ribosomes, limited availability of tRNA(Trp) (about 1% of total tRNA) facilitates a pause at the UGG codon (due to the vacant A site), allowing increased opportunity for ribosome realignment. Excess tRNA(Trp) reduces the time the A site is vacant and thus reduces the Frameshift. The slower hyperaccurate ribosomes increase the pause time and thus increase the opportunity for shifting, a process again reversed by increasing the in-frame cognate tRNA(Trp). These data provide strong support for a model in which the extent of ribosome pause time at a programmed Frameshift site is a major determinant in the efficiency of the Frameshift and in which tRNA availability can be a major influence on this process.
Robert L Duda - One of the best experts on this subject based on the ideXlab platform.
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a balanced ratio of proteins from gene g and Frameshift extended gene gt is required for phage lambda tail assembly
Journal of Molecular Biology, 2013Co-Authors: Roger W. Hendrix, Robert L DudaAbstract:In bacteriophage λ, the overlapping open reading frames G and T are expressed by a programmed Translational Frameshift similar to that of the gag-pol genes of many retroviruses to produce the proteins gpG and gpGT. An analogous Frameshift is widely conserved among other dsDNA tailed phages in their corresponding "G" and "GT" tail genes even in the absence of detectable sequence homology. The longer protein gpGT is known to be essential for tail assembly, but the requirement for the shorter gpG remained unclear because mutations in gene G affect both proteins. A plasmid system that can direct the efficient synthesis of tails was created and used to show that gpG and gpGT are both essential for correct tail assembly. Phage complementation assays under conditions where levels of plasmid-expressed gpG or gpGT could be altered independently revealed that the correct molar ratio of these two related proteins, normally determined by the efficiency of the Frameshift, is also crucial for efficient assembly of functional tails. Finally, the physical connection between the G and T domains of gpGT, a consequence of the Frameshift mechanism of protein expression, appears to be important for efficient tail assembly.
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conserved Translational Frameshift in dsdna bacteriophage tail assembly genes
Molecular Cell, 2004Co-Authors: Roger W. Hendrix, Robert L DudaAbstract:Abstract A programmed Translational Frameshift similar to Frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage λ, this Frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous −1 Frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous −2 Frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this Frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.
Juan E Suarez - One of the best experts on this subject based on the ideXlab platform.
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a 1 ribosomal Frameshift in the transcript that encodes the major head protein of bacteriophage a2 mediates biosynthesis of a second essential component of the capsid
Journal of Bacteriology, 2004Co-Authors: Pilar Garcia, Isabel Rodriguez, Juan E SuarezAbstract:A2 is a temperate bacteriophage that infects strains of several Lactobacillus casei-related species, including some used as probiotic components in fermented milks. It belongs to the family Siphoviridae, and its genome lies in a double-stranded DNA molecule 43,411 bp long which encodes 61 open reading frames (ORFs) and presents 3′-protruding cohesive ends (15). These may be grouped into several functional modules. Towards the center of the DNA lies the region that regulates the switch between the lytic and lysogenic cycles. Here, two adjacent, divergently oriented repressor genes, cI and cro, encode proteins that play roles superficially similar to those of the analogous proteins of bacteriophage lambda (14, 18-20). Downstream of cI is located the cassette that mediates integration of the phage DNA into the chromosome of its hosts (2), while cro is followed by the replication module of the phage (27). Downstream of this module there is a long DNA stretch that encodes a series of small polypeptides, most of which are unessential for development or lysogenization of the phage, at least under laboratory conditions (15, 20). This end of the genome is occupied by the small terminase subunit determinant, which marks the beginning of the morphogenetic region and is followed, towards the other side of the cohesive ends, by the large terminase subunit (13) and the genes that encode the structural components of the virion in the following order: capsid, head-tail connector, tail, and host recognition, all of which form a late-expressed, single operon (15). Finally, the host lysis cassette lies between the morphogenetic and integration modules. Analysis of the virion structural proteins revealed two polypeptides of 35 and 42 kDa (as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) that were the major components of the head at a ratio of 4:1. Surprisingly, both shared their amino termini, which matched an internal sequence of orf5 in the phage genome. In line with this finding, orf5 would originate two polypeptides of different sizes, both of which would suffer the same proteolytic processing upon incorporation into the capsid. This processing would render a hypothetical 123-amino-acid polypeptide from the amino end that is postulated to be the scaffolding protein of the phage and the two polypeptides of different sizes found in the virions (15). In this report, we confirm this hypothesis through presentation of data indicating that the small head protein, gp5A, corresponds to the translation product of orf5 after proteolytic processing, while its large counterpart, gp5B, results from a change in the frame of translation and is 85 amino acids longer than gp5A. Additionally, some requirements for the Frameshift to occur are revealed. Finally, it is demonstrated that both proteins are essential for the generation of viable phages. To our knowledge, this is the first report of a −1 Translational Frameshift occurring in a gram-positive-specific bacteriophage, although sequence analysis of the Listeria bacteriophage PSA suggests that this situation might be more general (36). In this recent study, two +1 Frameshifts in the major capsid and tail genes of PSA were reported.
Sherwood R Casjens - One of the best experts on this subject based on the ideXlab platform.
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A programmed Translational Frameshift is required for the synthesis of a bacteriophage λ tail assembly protein
Journal of Molecular Biology, 1993Co-Authors: Margaret E. Levin, Roger W. Hendrix, Sherwood R CasjensAbstract:Abstract Two proteins, one of 31 kDa and one of 16 kDa, are encoded by a segment of the phage λ tail gene region that contains two overlapping reading frames, neither of which is long enough to encode the larger protein. We show that the abundant 16-kDa protein (gpG) is encoded by the upstream open reading frame, gene G. The 31-kDa protein, gpG-T, is encoded jointly by gene G and the overlapping downstream T open reading frame. gpG-T is synthesized as the result of a Translational Frameshift that occurs when a ribosome translating the G gene slips back by one nucleotide at a position six codons from the C terminus of the gene and thereby bypasses the G termination codon to continue on in the T open reading frame. The resulting protein shares 135 residues of N-terminal amino acid sequence with gpG, followed by 144 amino acid residues of unique sequence. The Frameshift event occurs with a frequency of approximately 4% at the sequence G GGA AAG, which encodes the dipeptide -Gly-Lys in both the zero and -1 reading frames. The Frameshift frequencies of point mutants in this "slippery sequence" argue that codon-anticodon interactions with both the glycyl and the lysyl-tRNA are important for Frameshifting to occur. We find no clear evidence for a pausing mechanism to enhance Frameshifting, as is seen in other well-characterized Frameshifts. No simple secondary structure has been predicted for the region downstream from the slippery sequence, but this downstream sequence does contribute to the Frameshifting rate. Our results together with those of Katsura and Kuhl show that the Frameshift product, gpG-T, has an essential role in λ tail assembly, acting prior to tail shaft assembly. The role of gpG in tail assembly is not known. We find that both gpG and the gpG-T are absent from mature virions.
