Single‐particle cryo‐electron microscopy (cryo‐EM) became a well‐established method to study the structure and function of
large macromolecular assemblies in a close to physiological environment. Cryo‐EM reconstructions of ribosomal complexes trapped
at different stages during translation, cotranslational targeting, and translocation provide new insights on a molecular level
into these processes, which are vital for the correct localization and folding of all proteins in the cell. The EM structures
in combination with biochemical experiments and available high‐resolution crystal or nuclear magnetic resonance (NMR) structures
of individual factors and of the ribosome allow for interpretation in quasi‐atomic detail of the molecular mechanism of ribosomal
complexes, their conformational changes and dynamic interactions with factors like the signal recognition particle, SRP receptor,
the translocon, and the chaperone trigger factor. The snapshots obtained by single‐particle EM reconstructions enable us to
follow the path of a nascent protein from the peptidyl‐transferase center, through the ribosomal tunnel, to and across the
translocon in the membrane. With new developments in image processing techniques it is possible to sort a biological homogenous
sample into different conformational states and to reach subnanometer resolution such that folding of the nascent chain into
secondary structure elements can be directly visualized. With improved cryo‐electron tomography and correlative light microscopy
and EM, it will be possible to visualize ribosomal complexes in their cellular context. WIREs RNA 2012, 3:429–441. doi: 10.1002/wrna.119
The ribosomal tunnel directs initial folding of nascent chain (NC). (a) Cartoon drawing of the translating ribosome. The small and large ribosomal subunits (30S and 50S) are depicted in yellow and blue, respectively. The peptidyl‐transferase center (PTC) of 50S is occupied by P‐site tRNA (green). The nascent polypeptide (red) extends from the PTC into the ribosomal tunnel. Folding zones of the nascent polypeptide in the ribosomal tunnel are indicated. Folding of the nascent polypeptide has been reported in the upper and lower parts, but not in the constricted part of the tunnel. (b) Crystal structure of Escherichia coli 70S (PDB ID 2AVY and 2AW412) shown from the exit of the ribosomal tunnel with a view into the tunnel along the path of the NC. The tunnel wall mostly consists of ribosomal RNA (gray) with contributions of loops of ribosomal proteins L4 (purple), L22 (green) at the constriction, and L23 (orange) toward the exit.
Cotranslational folding in Escherichia coli. (a) Cartoon drawing of trigger factor (TF) chaperone action. TF binds to ribosomal protein L23 at the exit of the ribosomal tunnel and contacts all nascent chains (NCs) as soon as they emerge from the tunnel. The ribosome and TF provide the environment for the folding of cytoplasmic proteins (the cradle). TF can dissociate from the ribosome while still being connected with the folding NC. In the case of multi‐domain proteins, more than one TF can bind to the NC. (b) Crystal structure of E. coli TF (PDB ID 1W2626). The head domain of TF (yellow) has peptidyl–prolyl cis–trans isomerase activity, which is independent from TFs chaperone activity. The tail domain (red) and the arms (green and blue) form the cradle. (c) Cryo‐electron microscopy reconstruction of E. coli TF bound a ribosome displaying a folding NC (EMD‐149927). TF (red) binds to the large ribosomal subunit (blue), next to the exit of the ribosomal tunnel and blocks access of other proteins to the site where the NC emerges. Thereby, a protective environment is generated for folding of the NC.
Quasi‐atomic models of cotranslational targeting states. (a) In the docked signal recognition particle (SRP) state, the NG‐domain of Ffh (greenyellow) binds to ribosomal protein L23 (PDB ID 2J2835), the Ffh M‐domain (yellow) binds the signal sequence (ss, red) and the 4.5S RNA (orange). The signal sequence contacts the ribosome and the SRP. Four connections are formed between the SRP and the 50S. (b) In the early state, the FtsY NG‐domain (magenta) interacts with the RNA tetraloop and with the Ffh NG‐domain (PDB ID 2XKV36). The Ffh M‐domain forms the sole ribosomal contact, whereas SRP RNA and the Ffh NG‐domain are disconnected from the 50S as well as the M‐domain part, which contacts the signal sequence (not depicted).36 (c) The closed state has been modeled by aligning the SRP RNA of the SRP–FtsY crystal structure (PDB ID 2XXA37) onto the SRP RNA of the early state quasi‐atomic model (in b). In the closed state, the NG‐domains of Ffh and FtsY form a tight complex. Arrows in (a) and (b) indicate conformational changes that occur during the transition to the next targeting state.
Cryo‐electron microscopy (cryo‐EM) reconstructions along the pathway of Escherichia coli cotranslational targeting and translocation. The SRP binds with the Ffh NG‐domain next to the exit of the ribosomal tunnel and scans for the presence of an NC with a signal sequence. The M‐domain and 4.5S RNA are flexible in the scanning SRP. When SRP recognizes a signal anchor sequence, it fully docks onto the ribosome (docked SRP) (EMD‐125038) and adopts an elongated conformation stabilized by interactions with the 50S. The SRP RNA points away from the ribosomal tunnel exit, and the SRP RNA tetraloop is accessible. FtsY binding leads to formation of the early SRP–FtsY complex (EMD‐176236). Next to the exit of the ribosomal tunnel, additional V‐shaped density is visible. A cryo‐EM reconstruction of the closed or activated SRP/FtsY state is not available. After successful hand over of the translating ribosome, the SecYEG translocon binds to the exit of the ribosomal tunnel (EMD‐114314). The translocation channel is aligned with the ribosomal tunnel such that an almost continuous channel from the PTC into the periplasm is formed for the NC.
Protein export and membrane protein integration via the Sec translocation machinery. (a) SecYβγ crystal structure (PDB ID 1RHZ58) viewed from the cytoplasm. In the inactive state, the channel formed by SecY (green) is sealed by a small α‐helix, the plug. The homologs of SecE (purple) and SecG (brown) bind to SecY at the outside and do not contribute to the actual channel. (b) Quasi‐atomic model of a translocating protein conducting channel (PCC) (PDB IDs 3J00 and 3J01).63 The path of the nascent chain (NC) is indicated in red. The α‐helical signal sequence is integrated in the wall of the SecY resulting in an opening of the translocation pore. The remainder of the NC is translocated in the center of the channel in a mostly hydrophilic environment. Arrows indicate the opening of the SecY channel (green) and the possibility of the signal sequence to partition into the lipid bilayer (red). (c) Cryo‐electron microscopy reconstruction of the translating ribosome with a translocating nanodisc containing PCC (EMD‐1858),63 which was used for fitting of the quasi‐atomic model (model and color coding as in (b)). The map was clipped at the vertical plain to visualize the map‐model overlay of the translocating PCC. (d) RNC–translocon (model and color coding as in (b)) viewed from the plane of the membrane which is shown in gray. Cytoplasmic loops of SecY contact the 50S (cyan). The NC can enter directly into the translocation channel after emerging from the tunnel. The NC is translocated in a loop conformation in the center of the translocon.
and her research team focus on the dissection of the molecular mechanisms and pathways involved in Lin28-mediated regulation. First, they will analyze Lin28 expression in mouse and human ES cells to determine whether its expression is regulated during the cell cy-cle. Then, they will characterize the interactions between Lin28 and its associated mRNAs to gain molecular insights into their assembly, function and regulation in the cellular milieu. Finally, they will strive to identify Lin28-interacting protein partners and new target mRNAs to establish a comprehensive and global understanding of Lin28 function.