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Ribosome and ribosomal RNA Structure: An experimental and computational analysis of expansion segments in eukaryotic ribosomal RNA
Södertörns högskola, Institutionen för livsvetenskaper. Stockholms universitet, Wenner-Grens institut.
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Ribosomes are large ribonucleoprotein complexes which incorporate amino acids into peptide chains during translational process in all types of living cells. The eukaryotic ribosome is larger compared to its prokaryotic counterpart. The size differences are due to a larger protein part and that the rRNA contains eukaryote specific expansion segments (ES). Cryo-EM reconstruction has visualized many ES on the ribosomal surface which have given clues about function and structural features. However, the secondary structures of most ES are unknown or ill defined. In this thesis, the secondary and also to a certain extent the tertiary structures of several ES are determined by using computational methods and biochemical experimental techniques. The juxtaposition of ES6 close to ES3 in the Cryo-EM image of the yeast ribosome suggested that ES3 and ES6 might interact. A computational analysis of more than 2900 sequences shows that a complementary helical region of seven to nine contiguous base pairs can form between ES3 and ES6 in almost all analyzed sequences. Biochemical in situ experiments support the proposed interaction. Secondary structure models are presented for ES3 and ES6 in 18S rRNA and ES39 in 28S rRNA, where homologous structural elements could be modeled in the experimentally analyzed ribosomes from fungi, plants and mammals. The structure models were further supported by computational methods where the ES6 structure and the ES39 structure could be formed in more than 6000 and 900 sequences respectively. A tertiary structure model of ES3 and ES6 including the helical interaction is presented. An in vitro transcribed and folded ES6 sequence differed from that observed in situ, suggesting that chaperones, ribosomal proteins, and/or the tertiary rRNA interaction could be involved in the in vivo folding of ES6. An analysis of the similarities between ES39 structures suggests that it might be under selective constraint to preserve its secondary structure.

Ort, förlag, år, upplaga, sidor
Stockholm: Wenner-Gren Institute for Experimental Biology, Stockholm university , 2008. , s. 72
Nyckelord [en]
eukaryotes, expansion segment, ribosomes, rRNA, secondary structure, structure of rRNA.
Nationell ämneskategori
Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:sh:diva-32043ISBN: 978-91-7155-603-5 (tryckt)OAI: oai:DiVA.org:sh-32043DiVA, id: diva2:1073784
Disputation
2008-05-23, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-02-13 Skapad: 2017-02-13 Senast uppdaterad: 2018-01-13Bibliografiskt granskad
Delarbeten
1. A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits
Öppna denna publikation i ny flik eller fönster >>A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits
2003 (Engelska)Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 9, nr 1, s. 20-24Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:sh:diva-17371 (URN)10.1261/rna.2108203 (DOI)000183882300004 ()12554872 (PubMedID)2-s2.0-0037276155 (Scopus ID)
Tillgänglig från: 2012-11-22 Skapad: 2012-11-19 Senast uppdaterad: 2017-07-19Bibliografiskt granskad
2. Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits
Öppna denna publikation i ny flik eller fönster >>Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits
2004 (Engelska)Ingår i: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 10, nr 3, s. 403-411Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:sh:diva-17624 (URN)10.1261/rna.5135204 (DOI)000189115400008 ()14970386 (PubMedID)2-s2.0-10744227895 (Scopus ID)
Tillgänglig från: 2012-12-14 Skapad: 2012-12-14 Senast uppdaterad: 2017-07-19Bibliografiskt granskad
3. Analysis of the secondary structure of expansion segment 39 in ribosomes from fungi, plants and mammals
Öppna denna publikation i ny flik eller fönster >>Analysis of the secondary structure of expansion segment 39 in ribosomes from fungi, plants and mammals
2006 (Engelska)Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 357, nr 3, s. 904-916Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The structure of expansion segment 39, E539, in eukaryotic 23 S-like ribosomal RNA was analysed using a combination of chemical and enzymic reagents. Ribosomes were isolated from yeast, wheat, mouse, rat and rabbit, five organisms representing three different eukaryotic kingdoms. The isolated ribosomes were treated with structure-sensitive chemical and enzymic reagents and the modification patterns analysed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The expansion segment was relatively accessible to modification by both enzymic and chemical probes, suggesting that ES39 was exposed on the surface of the ribosomes. The collected modification data were used in secondary structure modelling of the expansion segment. Despite considerable variation in both sequence and length between organisms from different kingdoms, the structure analysis of the expansion segment gave rise to structural fingerprints that allowed identification of homologous structures in ES39 from fungi, plants and mammals. The homologous structures formed an initial helix and an invariant hairpin connected to the initial helix via a long single-stranded loop. The remaining part of the ES39 sequences accounted for most of the length variation seen between the analysed species. This part could form additional, albeit less similar, hairpins. A comparison of ES39 sequences from other fungi, plants and mammals showed that identical structures could be formed in these organisms.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:sh:diva-14303 (URN)10.1016/j.jmb.2006.01.043 (DOI)000236629300019 ()16473366 (PubMedID)2-s2.0-33644955476 (Scopus ID)
Tillgänglig från: 2011-12-20 Skapad: 2011-12-20 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
4. Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA
Öppna denna publikation i ny flik eller fönster >>Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA
2006 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, nr 26, s. 8067-8078Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.

Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:sh:diva-14288 (URN)10.1021/bi052149z (DOI)000238558700013 ()16800631 (PubMedID)2-s2.0-33745621079 (Scopus ID)
Tillgänglig från: 2011-12-20 Skapad: 2011-12-20 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
5. Comparative analysis of rRNA sequences from the large ribosomal subunit of more than 900 eukaryotic species reveals structural similarities in expansion segment ES39.
Öppna denna publikation i ny flik eller fönster >>Comparative analysis of rRNA sequences from the large ribosomal subunit of more than 900 eukaryotic species reveals structural similarities in expansion segment ES39.
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biologiska vetenskaper
Identifikatorer
urn:nbn:se:sh:diva-32044 (URN)
Anmärkning

Som manuskript i avhandling. As manuscript in dissertation.

Tillgänglig från: 2008-04-24 Skapad: 2017-02-13 Senast uppdaterad: 2017-02-13Bibliografiskt granskad

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