Identification and characterization of the ribosomal RNA operons from Rhodobacter sphaeroides
Dryden, Sylvia Cartwright
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https://hdl.handle.net/2142/23224
Description
Title
Identification and characterization of the ribosomal RNA operons from Rhodobacter sphaeroides
Author(s)
Dryden, Sylvia Cartwright
Issue Date
1992
Doctoral Committee Chair(s)
Kaplan, Samuel
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
The three ribosomal RNA operons present in the facultative photoheterotroph Rhodobacter sphaeroides were identified, cloned and sequenced. DNA sequence analysis has identified the 16S, 23S and 5S rRNAs, two tRNAs (ile and ala) in the 16S-23S spacer region, and an f-met tRNA immediately following the 5S rRNa gene of all three operons. The secondary structure of the rRNA and tRNA molecules present in the rRNA operons was deduced and evolutionary implications discussed. Physical mapping, genetic analysis, and Southern hybridization data indicate that rrnA is contained on a large chromosome and rrnB and rrnC are contained on a second smaller chromosome. Strains were constructed in which one or two rRNA operons were deleted and the effects on cell growth analyzed. Strains with deletions in one or two rrn operons grew more slowly than wild type strains under all growth conditions tested. However, there was no significant difference in the growth rate of strains with two rrn operons deleted when compared to strains deleted in only one operon. In addition, the primary transcript of each rRNA operon was identified via primer extension analysis. The region upstream of the primary rRNA transcripts was analyzed and a $-$10 and $-$35 promoter region was identified as well as regulatory elements which may be involved in regulating the synthesis of the rRNA operons. Fusions of the proposed promoter regions were constructed utilizing the reporter molecule xylE and analyzed under various growth conditions. Results indicate that production of the xylE gene product (catechol 2,3-dioxygenase) was greatest under photosynthetic conditions. Also, the upstream region of rrnB, when fused with xylE produced more catechol 2,3-dioxygenase than analogous regions of rrnA, suggesting that the promoters of the rrn operons differ in strength. It is postulated that under the same conditions, the rrnC promoter could potentially be the weakest of the three rrn promoters.
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