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Microbiology: Current Research 2017 | Volume 1, Issue 2
allied
academies
Joint Conference
GLOBAL APPLIED MICROBIOLOGY CONFERENCE
MICROBIAL & BIOCHEMICAL RESEARCH AND TECHNOLOGIES
&
October 18-19, 2017
Toronto, Canada
International Congress on
I
n prokaryotes two adjacent ORF are often linked with an
overlapping combination of termination and initiation codons
with a total of 4 or 5 nucleotides, for example UAAUG or AUGA.
In these junctions, ribosomes at the stop codon are released
by RRF and some of them re-bind to the nearby AUG and start
translating the downstream ORF. In the absence of RRF, the
ribosome at the stop codon remains on the mRNA and would
read it in framewith the termination triplet. We studied the role
of RRF in these junctions
in vitro
and
in vivo
. For
in vivo
studies,
we used an
E. coli
strain with a temperature sensitive mutation
of RRF, so that we could inactivate the function of RRF at the
non-permissive temperature (39°C). We show that for correct
reading of the downstream ORF, AUG is essential. The shorter
the upstream ORF the lower will be the downstream reading.
Introduction of a complementary sequence to the 3’-terminal
regions of 16S rRNA into the mRNA increased downstream
reading fromAUGA. Shorteningof theupstreamORF to4codons
completely abolished the downstream reading of UAAUG.
This suggests that if Shine-Dalgarno (SD) sequence is near the
termination codon, the RRF-released ribosome is attracted by
the SD sequence to the extent that it loses the downstream
movement after it is released. For
in vitro
studies, we used
the PURE system, so that we could omit RRF in the reaction
mixture. We have confirmed,
in vitro
, the essence of the
in vivo
observation described above using amRNAhaving the following
sequence: “GGGAAUUCAAAAAUUUAAACAGGUAUACAUACU
AUG UUU ACG AUU ACU ACG AUC UUC UUU ACG AUC UUC
UUU ACG AUU ACU ACG AUC UUC UUU ACG AUU ACU ACG
AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG UAAUG
CGU CUG CAG GCA UGC AAG CUA A24A” (Bold character is
the junction sequence broken underline is the Shine-Dalgarno
sequence). In the presence of RRF, the downstream reading
starts from AUG causing the incorporation of [14C]-Leucine
(CUG and CUA). On the other hand in the absence of RRF, the
first triplet read was UGC of UAAUG CGUC and [14C]-Valine was
incorporated due to the codon GUC. Upstream reading was
detected by [3H]-phenylalanine incorporation (due to UUUs
and UUCs). With this assay, we also showed that Fusidic Acid
could inhibit RRF at lower concentration than that necessary to
inhibit translocation (monitored by the incorporation of [3H]-
phenylalanine), causing the inhibition of the incorporation
of [14C]-Leucine. Moreover, using ribosome with tethered
subunits (1), we were able to show that the splitting of the
ribosomal subunits was not necessary in the recycling reaction,
demonstrating that recycling of the ribosome take place also in
the absence of the splitting of the ribosomal subunits.
Speaker Biography
Akira Kaji is a Professor of Microbiology, School of Medicine, University of Pennsylvania.
He has contributed to the deciphering of genetic code by his discovery of the fact
that the complex of poly-U with ribosome binds specifically to tRNA specific for
phenylalanine. He also discovered RRF.
e:
kaji@pennmedicine.upenn.eduAkira Kaji
University of Pennsylvania, USA
In vivo
and
in vitro
studies of RRF (ribosome recycling factor) revealed that its major
function is to release mRNA from the post-termination complex and not splitting of
the ribosomal subunits