@article{juzumiene_wollenzien_2001, title={Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking}, volume={7}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838201001728}, abstractNote={The 16S rRNA central pseudoknot region in the 30S ribosomal subunit has been investigated by photocrosslinking from 4-thiouridine (s4U) located in the first 20 nt of the 16S rRNA. RNA fragments (nt 1-20) were made by in vitro transcription to incorporate s4U at every uridine position or were made by chemical synthesis to incorporate s4U into one of the uridine positions at +5, +14, +17, or +20. These were ligated to RNA containing nt 21-1542 of the 16S rRNA sequence and, after gel purification, the ligated RNA was reconstituted into 30S subunits. Long-range intramolecular crosslinks were produced by near-UV irradiation; these were separated by gel electrophoresis and analyzed by reverse transcription reactions. A number of crosslinks are made in each of the constructs, which must reflect the structural flexibility or conformational heterogeneity in this part of the 30S subunit. All of the constructs show crosslinking to the 559-562, 570-571, and 1080-1082 regions; however, other sites are crosslinked specifically from each s4U position. The most distinctive crosslinking sites are: 341-343 and 911-917 for s4U(+5); 903-904 (very strong), 1390-1397, and 1492 for s4U(+14); and 903-904 (moderate) for s4U(+17); in the 1070-1170 region in which there are different patterns for each s4U position. These results indicate that part of the central pseudoknot is in close contact with the decoding region, with helix 27 in the 885-912 interval and with part of domain III RNA. Crosslinking between s4U(+14) and 1395-1397 is consistent with base pairing at U14-A1398.}, number={1}, journal={RNA}, author={Juzumiene, DI and Wollenzien, P}, year={2001}, month={Jan}, pages={71–84} }
 @article{juzumiene_shapkina_kirillov_wollenzien_2001, title={Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions}, volume={25}, ISSN={["1046-2023"]}, DOI={10.1006/meth.2001.1245}, abstractNote={We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.}, number={3}, journal={METHODS}, author={Juzumiene, D and Shapkina, T and Kirillov, S and Wollenzien, P}, year={2001}, month={Nov}, pages={333–343} }
 @article{juzumiene_wollenzien_2000, title={Organization of the 16S rRNA around its 5 ' terminus determined by photochemical crosslinking in the 30S ribosomal subunit}, volume={6}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838200991659}, abstractNote={The organization of the 5' terminus region in the 16S rRNA was investigated using a series of RNA constructs in which the 5' terminus was extended by 5 nt or was shortened to give RNA molecules that started at positions -5, +1, +5, +8, +14, or +21. The structural and functional effects of the 5' extension/truncations were determined after the RNAs were reconstituted. 30S subunits containing 16S rRNA with 5' termini at -5, +1, +5, +8 and +14 had similar structures (judged by UV-induced crosslinking) and exhibited a gradual reduction in tRNA binding activity compared to that seen with 30S subunits reconstituted with native 16S rRNA. To create the 5' terminal site-specific photocrosslinking agent, the reagent azidophenacylbromide (APAB) was attached to the 5' terminus of 16S rRNA through a guanosine monophosphorothioate and the APA-16S rRNAs were reconstituted. Crosslinking carried out with the APA revealed sites in six regions around positions 300-340, 560, 900, 1080, the 16S rRNA decoding region, and at 1330. Differences in the pattern and efficiency of crosslinking for the different constructs allow distance estimates for the crosslinked sites from nucleotide G9. These measurements provide constraints for the arrangement of the RNA elements in the 30S subunit. Similar experiments carried out in the 70S ribosome resulted in a five- to tenfold lower frequency of crosslinking. This is most likely due to a repositioning of the 5' terminus upon subunit association.}, number={1}, journal={RNA}, author={Juzumiene, DI and Wollenzien, P}, year={2000}, month={Jan}, pages={26–40} }