Protein Targeting
And the Signal Recognition
Particle (SRP)
Background
• Peptide growth begins at the N terminus
• There are 2 types of ribosomes:
-those that are free in the cytosol
-those that are bound to the endoplasmic reticulum
• Transmembrane and secretory proteins are synthesized
by Ribosomes bound to the ER
• Targeting pathways process approximately 40% of all
proteins
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Signal Recognition Particle
• What are Signal Recognition Particles?
– SRPs are protein-RNA complexes
– They are responsible for the targeting of membrane
proteins
– These proteins are universal among all kingdoms of
life
Prokaryotic vs. Eukaryotic SRP
• Eukaryotic SRPs consist of 6 polypeptides (SRP9,
SRP14, SRP19, SRP54, SRP68, SRP72) and a 7S RNA
– Human 7S RNA is 299nt long and EM has shown it to
be rod shaped
• Prokaryotic SRPs are smaller
– E. coli SRPs have only one polypeptide called Ffh
(fifty-fourth homologue)
– This protein is 48kDa
– Has a 4.5S RNA strand (Ffs)
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Prokaryotic vs. Eukaryotic SRP
• Prokaryotic Ffh and Ffs are highly conserved
• They are a minimal functional version of the SRP complex
• In Vitro, Ffh and Ffs can be used to replace SRP54 and 7S RNA in
Eukaryotic SRPs
– SRP will still maintain its function
• Eukaryotic RNA has 4 domains, of which only domain IV is
conserved
– Tetraloop
– Asymmetric Loop
– Symmetric Loop
Comparison
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E. Coli SRP superimposed on
Mammalian SRP Domain
Experimental Procedure
• Generated a nascent chain containing a transmembrane
helix (TMH) of E. coli FtsQ (a membrane protein)
• This formed a stable ribosomal nascent chain complex (
RNC)
• Purified RNCs by a sucrose gradient centrifugation and
affinity chromatography
• Almost all RNCs were in complex with SRPs under
conditions for electron microscopy (EM)
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Cryo-EM
• Structural Determination was done using Cryo-EM
• RNC-SRP complex had a resolution of 16Ǻ
• 70S-SRP complex had a resolution of 20Ǻ
Cryo-EM Structure of RNC
• Halic et al. found an extra density at the ribosome exit
tunnel was observed that is not present in ribosomes
without the nascent chain
• Concluded that this density was the result of an
emerging nascent chain
• Emerging sequence must be in the form of an α helix
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Cryo-EM of RNC-SRP Complex
• Blue – 50S Ribosomal
Subunit
• Yellow – 30S Ribosomal
Subunit
• Pink – SRP
• Star – Polypeptide exit
tunnel
Assigning a Structure to SRP
• Modeled SRP using:
– NG Domain of Thermus aquaticus
– M Domain of Sulfolobus solfataricus (in complex with
4.5S RNA)
– Energy minimization
• Cryo-EM structure was different than previously
calculated structure via X-ray crystallography
• Cryo-EM structure was elongated due to flexible regions
– Linker Helix located between the M and NG domains
– Flexible hinge region in the G domain
• Possible explanation- conformational changes upon
binding to the ribosome
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SRP Structure
• Ffh and SRP 54 have 3 domains – N,G &M
• N terminal domain mediates the binding of the ribosome
– N domain (N-terminal domain) consists of a bundle of
4 antiparallel α helices
• The G domain has GTPase function
– Open β sheet which resembles other GTPases (RASlike)
• N and G domains are coupled in both structure and
function
• The M domain is the (C -terminal domain)
– It is rich in met residues (14 0f 102 residues in E. coli)
– Functions in binding the Ffs as well as the signal
peptide
– Has a hydrophobic binding pocket
• Cryo-EM structure – Left
• X-ray Structure – Right
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Cryo-EM of RNC-SRP Complex
• At the exit tunnel a large density is observed. This is
SRP
• Because of conformational heterogeneity of SRP the
resolution for SRP is lower than the ribosome
• 4.5S RNA is an elongated portion with a helical twist
• M Domain of SRP is seen next to the 4.5S RNA
• N and G Domains are barely visible at this resolution
Differences Observed Between
Prokaryotic and Eukaryotic SRP
• Ffh does not cover the exit tunnel of the prokaryotic
ribosome
• This allows other factors to come into contact with the
nascent chain
• SRP54 covers the exit tunnel
• SRP 54 have an Alu domain which stalls translation by
preventing the binding of elongation-factors
• Ffh does not have this domain
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Stereo View of SRP
•
•
•
•
NG Domain –Green
M Domain – Yellow
4.5S RNA – Orange
TMH - Red
4.5 S RNA
• Has a secondary
structure that is similar to
Domain IV
• Forms a 70 Å long rodshaped double helix
• Has a 4nt unpaired
tetraloop which enables
the RNA chain to double
back on itself
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4 Contact Sites
• There are 4 contact sites
between the large
ribosomal subunit and the
SRP (c1-c4)
• Of the 4, C1, C2, and C4
are conserved between
Ffh and Eukaryotic SRP
C1 Binding Site
• N Domain of Ffh
– Main contact site is L23 of the Ribosome
– Contacts L29 to a smaller degree
• C1 is conserved in SRP54
• Eukaryote N Domain has the same 2 contact sites with
the ribosome
• N Domain has 3 helices (h1-h3) which form the contact
site
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C1 Binding Site
• Ionic interactions between L23 and the N Domain
establish the binding site
• Red-Basic Residues
Blue-Acidic Residues
C1 Binding Site
• In Eukaryotic SRP54 contact at the C1 site is formed so
that the NG is perpendicular to the ribosome’s surface
• However in Ffh, L23 is contacted by the helices of the N
domain. This positions the G Domain into the vicinity of
L29
– Results in a different positioning of the NG and M
Domains than Eukaryotic SRP
– Places the M and G domains into close proximity
– points the M domain away from the N Domain
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Right – Eukaryote
Left – E. coli
Discrepancies??
• Study by Halic et.al used
X-ray crystallography
data to assign structure to
SRP density
• Found that the NG
domain was
perpendicular to the
ribosome surface, just as
in eukaryotic SRP
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C2 Binding Site
• Involves helix 24 of 23S RNA and L24 and L22 of the
50S ribosome subunit
• Also involves helix M5 of the M domain and possibly
helices M3 and M4
C3 Binding Site
• Only binding site that is
not well conserved
between Prokaryotic and
Eukaryotic SRP
• Binding site that is
furthest from the
polypeptide tunnel exit
• Involves nucleotides 7276 of 4.5S RNA which are
part of the kink region
and L18 (Green)
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C4 Binding Site
• A pronounced
conformational change in
helix 59 of the 23S RNA
is observed
(gray→green)
– 9Ǻ movement towards
the SRP M Domain
(yellow)
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M Domain
• Has a deep groove located
near the exit site which
binds to the nascent chain
• Groove is almost completely
lined with hydrophobic
residues (contains 11 of 14
Met residues)
M Domain
• In the absence of a
nascent chain the N
domain of Ffh binds to
the ribosome at the C1
binding site
• In the Cryo-EM structure
of 70S SRP complex
SRP density is seen only
at the C1 binding site
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M Domain
• The M domain is then
able to scan the growing
polypeptide chain for a
signal sequence
• Once a signal sequence
is found, the SRP
molecule is able to bind
fully at the other 3 binding
sites
Protein Targeting
• Proteins are synthesized with a signal region that is 1336 residues long
• This region consists of a 6-15 residues hydrophobic core
• This core is flanked by hydrophilic residues that often
have a basic residue close to the N terminus
• When the signal sequence protrudes beyond the
ribosome it is bound to by a Signal Recognition Particle
(SRP)
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Protein Targeting
• The SRP-Ribosome complex moves to the RER surface
where it is bound by a SRP receptor which is in complex
with a translocon (Sec61 in Eukaryotes and SecYEG in
Prokaryotes)
– GDP that is bound to the SR is replaced by GTP
• Upon binding the SRP and SR stimulate each other to
hydrolyze their GTP to GDP. This causes
conformational changes leading to the dissociation of
SRP from the SR
– Ribosome-Translocon complex is formed
Protein Targeting
• The now bound ribosome is able to resume polypeptide
synthesis with the N-terminal of the growing polypeptide
chain passing through the translocon into the lumen of
the ER
• Once the signal sequence has entered into the ER it is
cleaved from the polypeptide chain by a signal peptidase
• The polypeptide then begins to fold with the aid of
chaperones
• After polypeptide synthesis is complete it is released
from the Ribosome-translocon complex.
– The Ribosome dissociates from the RER and the
process begins again
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Protein Targeting
Driessen et al NSB 2001
GTP Hydrolysis Drives the Pathway
• GTPase functions of SRP54 and the SR mediate
targeting of the Ribosome-SRP complex
• GTPase Activating Protein (GAP) is required for GTP
hydrolysis
• Guanine Nucleotide Exchange Factor (GEF) is required
for exchange of GDP for GTP
• The complex formed between the M domain and the
signal sequence acts as the GEF for the SRP
– Causes the G Domain to exchange its GDP for GTP
– Followed by a conformational change that locks the
SRP and the Ribosome together and stops translation
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GTP Hydrolysis Drives the Pathway
• The empty translocon acts as a GEF for the SR
• The translocon associates with the SR and then the
SRP-Ribosome complex binds
• The SRP and SR complexes act as mutual GAPs
– Each stimulates the GTPase functions of the other
• GTP hydrolysis results in another conformational
change, causing the dissociation of the SRP complex
An Alternative Pathway
Driessen et al NSB 2001
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