| 1. |
- Antoun, Ayman, et al.
(författare)
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How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis
- 2006
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 23:2, s. 183-193
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Tidskriftsartikel (refereegranskat)abstract
- During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNA(fMet) bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.
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| 2. |
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| 3. |
- Darfeuille, Fabien, et al.
(författare)
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An antisense RNA inhibits translation by competing with standby ribosomes
- 2007
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 26:3, s. 381-392
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Tidskriftsartikel (refereegranskat)abstract
- Most antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an antisense RNA inhibits translation without binding directly to a TIR. The tisAB locus encodes an SOS-induced toxin, and IstR-1 is the antisense RNA that counteracts toxicity. We show that full-length tisAB mRNA (+1) is translationally inactive and endonucleolytic processing produces an active mRNA (+42). IstR-1 binding inhibits translation of this mRNA, and subsequent RNase III cleavage generates a truncated, inactive mRNA (+106). In vitro translation, toeprinting, and structure mapping suggest that active, but not inactive, tisAB mRNAs contain an upstream ribosome loading or “standby” site. Standby binding is required for initiation at the highly structured tisB TIR. This may involve ribosome sliding to a transiently open tisB TIR. IstR-1 competes with ribosomes by base pairing to the standby site located 100 nucleotides upstream.
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| 4. |
- Dunleavy, Elaine M., et al.
(författare)
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A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast Centromeres
- 2007
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 28:6, s. 1029-1044
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Tidskriftsartikel (refereegranskat)abstract
- A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(CnP1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.
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| 5. |
- Esberg, Anders, et al.
(författare)
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Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.
- 2006
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 24:1, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis, and formation of modified wobble uridines in tRNA. Therefore it was important to clarify whether Elongator has three distinct functions or whether it regulates one key process that leads to multiple downstream effects. Here, we show that the phenotypes of Elongator-deficient cells linking the complex to transcription and exocytosis are suppressed by increased expression of two tRNA species. Elongator is required for formation of the mcm(5) group of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) in these tRNAs. Hence, in cells with normal levels of these tRNAs, presence of mcm(5)s(2)U is crucial for posttranscriptional expression of gene products important in transcription and exocytosis. Our results indicate that the physiologically relevant function of the evolutionary-conserved Elongator complex is in formation of modified nucleosides in tRNAs.
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| 6. |
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| 7. |
- Johansson, Magnus, et al.
(författare)
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The kinetics of ribosomal peptidyl transfer revisited
- 2008
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 30:5, s. 589-598
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Tidskriftsartikel (refereegranskat)abstract
- The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.
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| 8. |
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| 9. |
- Pandey, Radha Raman, et al.
(författare)
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Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation
- 2008
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 32:2, s. 232-46
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Tidskriftsartikel (refereegranskat)abstract
- Recent investigations have implicated long antisense noncoding RNAs in the epigenetic regulation of chromosomal domains. Here we show that Kcnq1ot1 is an RNA polymerase II-encoded, 91 kb-long, moderately stable nuclear transcript and that its stability is important for bidirectional silencing of genes in the Kcnq1 domain. Kcnq1ot1 interacts with chromatin and with the H3K9- and H3K27-specific histone methyltransferases G9a and the PRC2 complex in a lineage-specific manner. This interaction correlates with the presence of extended regions of chromatin enriched with H3K9me3 and H3K27me3 in the Kcnq1 domain in placenta, whereas fetal liver lacks both chromatin interactions and heterochromatin structures. In addition, the Kcnq1 domain is more often found in contact with the nucleolar compartment in placenta than in liver. Taken together, our data describe a mechanism whereby Kcnq1ot1 establishes lineage-specific transcriptional silencing patterns through recruitment of chromatin remodeling complexes and maintenance of these patterns through subsequent cell divisions occurs via targeting the associated regions to the perinucleolar compartment.
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| 10. |
- Trobro, Stefan, et al.
(författare)
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A Model for How Ribosomal Release Factors Induce Peptidyl-tRNA Cleavage in Termination of Protein Synthesis
- 2007
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765 .- 1097-4164. ; 27:5, s. 758-766
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Tidskriftsartikel (refereegranskat)abstract
- A major unresolved question in messenger RNA translation is how ribosomal release factors terminate protein synthesis. Class 1 release factors decode stop codons and trigger hydrolysis of the bond between the nascent polypeptide and tRNA some 75 Å away from the decoding site. While the gross features of the release factor-ribosome interaction have been revealed by low-resolution crystal structures, there is no information on the atomic level at either the decoding or peptidyl transfer center. We used extensive computer simulations, constrained by experimental data, to predict how bacterial release factors induce peptide dissociation from the ribosome. A distinct structural solution is presented for how the methylated Gln residue of the universally conserved GGQ release factor motif inserts into the ribosomal A site and promotes rapid reaction with the peptidyl-tRNA substrate. This model explains key mutation experiments and shows that the ribosomal peptidyl transfer center catalyzes its two chemical reactions by a common mechanism.
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