Summary
Short-chained RNA species amplified
by bacteriophage Qβ replicase
E. Avota
The form of the thesis is the summary of published or prepared scientific articles. Only those results, not
included in articles, are shown in separate part of the analysis of the results. The work is done in
Biomedical Research and Study Centre of Latvian University in the time period between 1991 and 1997
years. The part of experiments was done in Max-Planck Institute of Biophysical Chemistry (Gottingen,
Germany).
The minimal structure elements of small RNA necessary for succesfull replication by
Qβ replicase still are under the close investigation. To study the problem, attention was
paid to the well known autocatalyticaly replicating small SV-7 RNA. The recloning of
RQ135 cDNA (modified DNA copy of SV-7 small RNA) under the control of T7
promoter, site directed mutagenesis and template activity analysis of in vitro transcribed
mutant RNA are described in this thesis. Experimental approach was developed to
measure the replication activity of non-viral small RNAs in vitro in the presence of Qβ
phage RNA polymerase. The influence of secondary structure changes on the
replication activity was determined. The destroying of continious double-stranded stem
of SV-7 RNA as well as symmetry of plus and minus RNA chains lowers the
replication rate about half. So the mathematicaly calculated secondary structure variant
of SV7 RNA with high level of base pairing proved to be important in replication by
Qβ replicase. The additional transcription initiation site at 3'end of template RNA had
no influence on the correct start of replication at the transcription initiation site.
The other part of the thesis is devoted to the studies of 6S RNA origin in Qβ
infected cells. Population of small RNA replicating in Qβ phage infected bacteria E.coli
cells were obtained and analyzed in vitro for the first time since discovery of replicative
6S RNA in the infected cells. Novel Qua phage host cell RNA species are revealed to
be involved in the appearance of 6S RNA. Surprisingly, among the number of 6S
RNA precursors are fragments of bacteria E.coli cell RNAs: 23S rRNA, 10Sa RNA,
6S RNA and tRNA. The sequence complementary to the RNA fragment derived from
3518-3596 nucleotide positions of Qβ RNA genome also was among the other 6S
RNA species that was cloned. It shows the cabability to bind Q,8 polymerase and
replicate in vitro in the presence of Qua polymerase. This Qβ RNA related sequence
was not classified previously as phage replicase binding site or sequence necessary for
succesfull Qβ genome replication.
Isolated short 6S RNA species are low-efficiency templates for Qβ polymerase.
Their replication rates are 4-20 times lower than is measured for small RNA generated
and optimized in vitro by Qβ replicase. None of the autocatalytic RNAs replicating in
vitro by Qβ replicase are found in vivo in Qβ phage infected E.coli cells. Therefore the
assumption that non-viral replicating RNAs generated in vitro in template-free synthesis
are derived from 6S RNA species found in vivo is not correct. The mechanism of
small RNA generation in vivo and in vitro must be fundamentally different.
The quantitative analysis of the obtained cDNA clones shows 6S RNA as the
phenomenon with nonregular occurance. Its replication in the Qβ infected bacteria cells
is only the casual side event in the infection process. Short -chained replicable RNA is a
very minor part of the phage RNA synthesized in the infection cycle.
Although it was in small ammount and diversity, replicative small RNA fraction
was found and studied also in the recombinant Qβ polymerase overproducing cells.
Even in the cells expressing high amounts of Qβ replicase very few RNA species could
be isolated.
The results described in the thesis are published or in print in ISI journals: "Nucleic
Acids Research", "Journal of Molecular Biology" and in Springer Laboratory Manual
"Basic cloning Procedures". They are presented also in two international symposiums.