RNase E Poster AMGEN .pdf

INTRODUCTION:
The RNA degradosome is a multi-enzyme complex present in most bacteria that is involved in RNA metabolism, degradation of messenger
RNA, processing of structural RNA and, consequently, post-transcriptional control of gene expression. The complex consists of enzymes
RNase E, PNPase, RNA helicase B and metabolic enzymes Enolase in E. coli and Aconitase in C. crescentus.
The RNase E globular amino-terminal half specifies the endonuclease activity and RNase E is the primary catalytic component of the
complex. The carboxy-terminal half is predominantly unstructured and it functions as a macromolecular-interaction domain that contains
binding sites for other interacting enzymes of the RNA degradasome.

Figure 1. – Rnase E and RNA Degradasome Strucutres ([1] George A. Mackie. RNase E: at the interface of bacterial RNA processing and decay, Nature Reviews Microbiology 11, 45-57 )

OBJECTIVE:
 [1] Developing chromosomal fusion RNase E – mCherry – miniSOG for localisation and structural studies in E. coli
 [2] Characterising Aconitase in C. crescentus RNA degradasome

[1] RNase E – mCherry – miniSOG:
[A]: Using an E. coli strain
genetically tagged with mCherry
at Rnase E for miniSOG fusion

[2] ACONITASE:
[A]: Aconitase of C.
crescentus was
expressed in the E.
Coli

[A]
mCherry

[B]

[B]: The miniSOG protein is a
new genetically encoded tag
used for labeling the proteins
which can be targeted to
different cellular locations for
light and EM imaging.

[B]: The gel band
patterns of purified
fractions indicate the
possible existence of
monomeric and
dimeric forms of
aconitase

[C]
Primer A

miniSOG
+ STOP

Downstream
homology
region

KmR + STOP

Primer B

[C]: PCR fragments for linear
transformation consists
homologous sequences with
target region

PCR step 1
Primer C

[D]: The lambda red system
(plasmid pKD 46) allows efficient
recombination between short
homologous sequences. This
requires a high internal DNA
concentration, achievable by
electroporation.
[E]: Screening by PCR to check
for the correct fusion of
miniSOG and kanamycin
]
sequence in the target region [D]
of RNase E.

mCherry
homology
region

KmR + STOP

Downstream
homology
region

mCherry
homology
region

Downstream
homology
region

mCherry

5,6 nm

[C]: Results from
dynamic light
scattering support the
presence of both
monomeric and
dimeric forms of
[D]
Aconitase

PCR step 2

Mini
SOG
+
STOP

[B]

7,5 nm

[C]

Primer B
mCherry
homology
region

[A]

[D]: To reduce residual
impurities, aconitase is
fused with a HIS-TAG
sequence

Aconitase

Aconitase

Aconitase

[E]

Downstream region

Aconitase

PERSPECTIVE:

mCherry

Created fusion RNase –
mCherry – miniSOG might
provide insight in to cellular
localisation of complex and
RNA metabolism, as well
as it’s interactions with subcellular structures using
correlative fluorescent - EM
microscopy and
crystallographic studies

KmR + STOP

[F]
[E]

[E]: Presence of insert
was confirmed by gel
electrophoresis

miniSOG + STOP

Downstream homology region

3,0 Kb Chromosomal Fusion
mCherry

miniSOG
+ STOP

RNase E + mCherry + miniSOG

KmR + STOP

PERSPECTIVE:

Downstream
homology
region

Figure 2. – Developing steps of RNase – mCherry – miniSOG chromosomal fusion

His-tag purification process provides
highly efficient sample for crystallographic
studies Aconitase in RNA degradasome of
C. crescentus

Predrag Stojakovic
predragstojakovicmb@gmail.com
University of Belgrade, Serbia
Faculty of Pharmacy

Figure 3. – Towards characterization of Aconitase

[2] Kirill A. Datsenko, One-step inactivation of chromosomal genes in Escherichia
coli K-12 using PCR products
[3] Xiaokun Shu, A Genetically Encoded Tag for Correlated Light and Electron
Microscopy of Intact Cells, Tissues, and Organisms
[4] Kenan C. Murphy, PCR-mediated gene replacement in Escherichia coli

ACKNOWLIDGMENTS:
Thanks to AMGEN Foundation for
funding this research project