Ribosomes are multicomponent macromolecular particles and
are essential for the survival of cells in all organisms. The ribosome's
universal function is to catalyze polypeptide synthesis through
translation of mRNA transcripts. Ribosomes from Escherichia coli,
eubacterial organisms, have a sedimentation coefficient of 70S and are
composed of 30S and 50S ribonucleoprotein subunits. The small
ribosomal subunit is an assembly of 21 different proteins and a 16S
ribosomal RNA. Within the 16S rRNA there are a few short stretches of
universally conserved sequences spanning positions 517-533, 1394-1408,
and 1492-1506. Clear functions for these sequence zones have not yet
been assigned.
Here I report a kinetic analysis of these highly conserved regions
in the 16S rRNA and within the 30S ribosomal subunits. Binding affinity
was measured in experiments that were based on protection from
nuclease 51 digestion of short oligodeoxynucleotides hybridized to the
designated regions. DNAs hybridized to regions 1400 and 1500 show
significant differences in the apparent dissociation constants when
measured in 30S particles as opposed to those found for 16S rRNA.
Region 525 showed no difference in kinetic behavior.
To further elucidate the functional and structural role played by
the region centered about C1400 in 16S rRNA, a four nucleotide deletion
was constructed within this region. The deletion was introduced by
direct RNA manipulation using DNA/RNA hybridization, RNase H
digestions, and ligation of the correct RNA fragments with T4 RNA
ligase. I improved ligation efficiency of large RNA molecules by
including a connector looped short DNA oligomer. Recycling products
through phenyl boronate agarose (PBA-30) column also improved the
efficiency of ligation.
The mutagenized 16S rRNA fully reassembles into 30 particles
and the altered 30S subunit possesses all of the normal ribosomal
proteins. Altered ribosomes were functional in in vitro translation of
MS2 mRNA. The altered ribosomes have lower translational activity
relative to controls. Here I present indirect evidence suggesting that the
decrease in the synthesis of MS2 coat proteins is the result of premature
termination.
The altered 16S RNA in ribosomes had an apparent dissociation
constants with DNA probes comparable to those found for normal 16S
rRNA. This suggest that the RNA is less flexible in the particle relative
to normal 30S subunits. The deletion at 1400 did not have any effect on
the physical properties of the 1500 region, as measured by DNA
hybridization. A minor, but significant, effect on the 525 region was
observed. A possible RNA/RNA interaction within the 30S particle is
proposed to account for this observation. / Graduation date: 1992
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/36814 |
| Date | 10 September 1991 |
| Creators | Almehdi, Mirza A. |
| Contributors | Schaup, Henry W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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