Download RNA-protein interaction

RNA-protein interaction
Students: Guo Xiaoyong
Fan Wenzhu Liu Yunhui
一.Background
DNA
transcription
proteins
RNA
translation
proteins
proteins
RNA and proteins are two kinds of
most important molecules in cells.
The interaction between proteins
and RNA is one of the key issues
in molecular biology.
RNA, in its varied forms,interacts with
proteins to carry out fundamental roles
in the cell.
(2) mRNA、 rRNA & tRNA in translation
Translation proceed in cytoplasm
in an ordered process .It includes
three phages .It requires free amino acids,free energy,mRNA,tRNA,
Ribosomes and nonribosomal factors(eIF in Eukaryotes and IF in
some prokaryotes).It should be noted that the polypepetide sequence
is in total agreement with gene code since tRNA anticodons are complementary of mRNA codons and
the mRNA sequence is a mirror of
the gene sequence.
(4) sRNA and protein
Small regulatory RNAs, including siRNA ,
miRNA ,piRNA ,and hsRNA .commonly
referred to as RNA silencing, such as RNA
interference (RNAi), translational repressi
-on, and heterochromatin formation in all
higher eukaryotes and play important roles
in cellular processes as diverse as development,stress response, or transposon silencing. Soon after the discovery of small
regulatory RNAs, members of the RNase III
familyand Argonaute protein family, some
RNA binding proteins, and etc, were identified
as their major cellular protein interactors and
involved in the biogenesis and various cellular
functions of small RNAs.
Understanding the contributions of various
RNA to the control of translation (protein
synthesis on the ribosome) in the cell forms
an important theme within the structural
biology and biophysics group.
二. Outlook and further perspectives
RNA-protein interactions are a central
component of posttranscriptional regulation
at multiple levels including RNA processing,
transport and translation. The sequenced
Human genome reveals hundreds of potential
RNA binding proteins . A critical step towards
understanding the function of RNA binding
proteins is to identify and determine how they
interact with their target RNAs.
三. The article
1.Introduction
Telomerase is an essential cellular
ribonucleoprotein that solves the end
replication problem and maintains
chromosome stability by adding
telomeric DNA to the termini of
linear chromosomes . Here we use a
single molecule approach to dissect
the individual assembly steps of
telomerase.
Direct observation of complex formation
in real time revealed two sequential steps of
protein-induced RNA folding, establishing
a hierarchical RNP assembly mechanism:
interaction with the telomerase holoenzyme
protein p65 induces structural rearrangement
of telomerase RNA, which in turn directs the
binding of the telomerase reverse criptase to
form the functional ternary complex.
2.Methods
(1) Fluorescence resonance energy transfer (FRET)
FRET is a process in which an excited fluorophore (the donor)
transfers its excited state energy to a light absorbing molecule
(the acceptor). This transfer of energy is non-radiative, due to
a dipole-dipole interaction between the donor and acceptor.
(2) Construction of FRET-labeled telomerase RNA
by DNA-splinted RNA ligation
Cy3
Cy5
Cy5
Cy3
3.Results & Analysis
Full-length telomeraseRNA
labelled with FRET donor (Cy3)
and acceptor (Cy5). RNA
molecules wereimmobilized on
a streptavidin-coated surface by
a biotin molecule engineered
onto an extension of stem II. The
interaction sites with p65 (red)
and TERT (green) are highlighted.
FRET histograms of RNA molecules in the absence
ofprotein (grey bars),the presence of 10nM p65 (red
bars) or 10nM p65 plus32nM TERT1–516
(green bars).
By real-time observating ,the author
found that addition of purified p65
give rise to a second population.p65
did not alter fluorescence intensities
from the RNA singly labelled with
Cy3 or Cy5 at the same locations.
To investigate the assembly of p65–RNA–
TERT ternary complex,the authours used
a purified TERT polypeptide containing the
amino-terminal 516 amino acids.
A single-molecule FRET trajectory showing hierarchical RNP assembly
after the addition (black arrow) of a protein mixture of p65 (10 nM) and
TERT1–516 (10 nM), characterized by a p65-induced FRET change (red
arrow) followed by a second FRET transition after complete assembly of
the ternary p65–RNA–TERT1–516 complex (green arrow).
These results indicate RNP’S assemblye
is initiated by p65 binding, stabilizing
a RNA structural intermediate, which
in turn promotes thefunctional co-assembly
of TERT with telomerase RNA.
GA
To characterize the p65-induced assembly
intermediate structurally,the authors generated
a series of truncated constructs composed of
telomerase RNA stems I and IV.
From the above charter,the authors
deduce that the p65-induced RNA
conformational change occurs within
stem IV and requires the central stem
IV GA bulge, which is conserved across
all Tetrahymena species.
4.Conclusion
These experiments show a hierarchical assembly
mechanism for telomerase RNP in which the
protein subunits mould a specific RNA tertiary
structure in a stepwise fashion.The protein p65
induces a structural change within the stem IV
region of the RNA.The RNA conformation in
the p65–RNA complex is further altered by
binding of TERT, resulting in a compact RNA
tertiary fold within the functional telomerase
RNP.