Translation
in Four Dimensions
Protein Synthesis at Atomic
Resolution: Mechanistics of Translation in the Light of Highly Resolved Structures for the Ribosome Pp.1-53
Initiation and Inhibition of Protein Biosynthesis –
Studies at High Resolution Pp.55-65
High-Resolution Structures of Large Ribosomal Subunits
from Mesophilic Eubacteria and Halophilic Archaea at Various Functional States Pp.67-78
Three-Dimensional Electron Cryomicroscopy of Ribosomes Pp.79-91
Structure
and Function of the Acidic Ribosomal Stalk Proteins Pp.93-106
Structure and Function of Bacterial Initiation Factors Pp.107-119
Inhibitory Mechanisms of Antibiotics Targeting Elongation
Factor Tu Pp.121-131
Is tRNA Binding or tRNA Mimicry Mandatory for Translation
Factors? Pp.133-141
Protein Factors Mediating Selenoprotein Synthesis Pp.143-151
Back to top] Protein Synthesis at Atomic Resolution: Mechanistics of Translation in the Light of
Highly Resolved Structures for the Ribosome
Our understanding of the process
of translation has progressed rapidly since the availability of highly resolved
structures for the ribosome. A wealth of information has emerged in terms of
both RNA and protein structure and the interplay between them. This has
prompted us to revisit the astonishing “treasure trove” of functional data
regarding the ribosome that has accumulated over the past decades. Here we try
a systematic synopsis of these ribosomal functions in light of the
cryo-electron microscopic structures (resolution >7 Å) and the atomic x-ray
structures (>2.4 Å) of the ribosome.
Back to top] Initiation and Inhibition of Protein
Biosynthesis – Studies at High Resolution
Analysis of the high resolution
structure of the small subunit from Thermus thermophilus shed light on its
inherent conformational variability and indicated an interconnected network of
features allowing concerted movements during translocation. It also showed that conformational
rearrangements may be involved in subunit association and that a latch-like
movement guarantees processivity and ensures maximized fidelity. Conformational
mobility is associated with the binding and the anti association function of
initiation factor 3, and antibiotics interfering with prevent the initiation of
the biosynthetic process. Proteins
stabilize the structure mainly by their long basic extensions that penetrate
into the ribosomal RNA. When pointing into the solution, these extensions may
have functional roles in binding of
non-ribosomal factors participating in the process of protein biosynthesis. In
addition, although the decoding center is formed of RNA, proteins seem to serve
ancillary functions such as stabilizing ist required conformation and assisting
the directionality of the translocation.
Back to top] High-Resolution Structures of Large Ribosomal Subunits from Mesophilic
Eubacteria and Halophilic Archaea at Various Functional
States
Structural analysis of the
recently determined high resolution structures of the small and the large
ribosomal subunits from three bacterial sources, assisted by the medium
resolution structure of a complex of the entire ribosome with three tRNAs, led
to a quantum jump in our understanding of the process of the translation of the
genetic code into proteins. Results of these studies highlighted dynamic
aspects of protein biosynthesis; illuminated the modes of action of several
antibiotics; indicated strategies adopted by ribosomes for maximizing their
functional activity and revealed a wealth of architectural elements, including
long tails of proteins penetrating the particle’s cores and stabilizing the
intricate folds of the RNA chains. Binding of substrate analogues showed that
the decoding and the peptide-bond formation are accomplished mainly by RNA.
However, several proteins may be functionally relevant in directing the mRNA
and in mediating the proper orientation of the tRNA molecules within the
ribosomal rRNA frame. Elements involved in intersubunit contacts or in
substrate binding are inherently flexible, but maintain well-ordered
characteristic conformations in unbound particles. The ribosomes utilize this
conformational variability for optimizing their efficiency and minimizing
non-productive interactions, hence disorder of functionally relevant features
may be linked to less active conformations or to far from physiological
conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In
the small subunit they affect the decoding accuracy or limit conformational
mobility and in the large subunit they either interfere with substrate binding,
by interacting with components of the peptidyl transferase cavity, or hinder
the progression of the growing peptide chain.
Back to top]
Three-Dimensional Electron
Cryomicroscopy of Ribosomes
Single particle electron
cryomicroscopy is nowadays routinely used to generate three-dimensional
structural information of ribosomal complexes without the need of
crystallization. A large number of structures of functional important ribosomal
complexes have thus been determined using this technique. In E. coli 70S
ribosomes all three tRNA binding sites could be localized. The ternary complex
of EF-Tu·tRNA·GTP that delivers the tRNA to the ribosome was directly
visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three
different functional states of translocation have been studied and the
respective EF-G binding sites have been mapped. The level of resolution
achievable with electron cryomicroscopy allows conformational changes in the
domain structures of elongation factors to be modelled in terms of rigid body
movements. Structural information on eukaryotic ribosomes is also available for
yeast and mammalian 80S ribosomes. The structural differences between rabbit
80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA
expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped
analysing the structure of an 80S·EF2·sodarin complex and most recently the
binding of a hepatitis C virus IRES element to a yeast 40S subunit has been
studied. The first electron cryomicroscopical 3D reconstructions have further
been used to overcome the initial phasing problems in X-ray crystallographic
studies of the ribosome facilitating structure determination of the recent
atomic resolution structures of the 30S and 50S ribosomal subunits. In turn,
the knowledge of the atomic structure of the ribosome makes detailed
interpretations of cryo-EM maps possible at ~20 Å resolution.
Back to top] Structure and Function of the Acidic Ribosomal Stalk
Proteins
The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified.
GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a ‘landing platform’ for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown.
While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 – EF-G and P-protein – EF-2 couples.
The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an a-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The physiological role of the other dimerization mode is unclear and is in apparent contradiction to distances measured by fluorescence resonance energy transfer. The discrepancies between the crystal structures and results from other physico-chemical methods may partly be a consequence of the dynamic functions of the proteins, necessitating a high flexibility.
Back to top] Structure and Function of Bacterial Initiation
Factors
Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors.
Back to top] Inhibitory Mechanisms of Antibiotics Targeting
Elongation Factor Tu
Since the pioneering discovery of
the inhibitory effects of kirromycin on bacterial elongation factor Tu (EF-Tu)
more than 25 years ago [1], a great wealth of biological data has accumulated
concerning protein biosynthesis inhibitors specific for EF-Tu. With the
subsequent discovery of over two dozen naturally occurring EF-Tu inhibitors
belonging to four different subclasses, EF-Tu has blossomed into an appealing
antimicrobial target for rational drug discovery efforts. Very recently,
independent crystal structure determinations of EF-Tu in complex with two
potent antibiotics, aurodox and GE2270A, have provided structural explanations
for the mode of action of these two compounds, and have set the foundation for
the design of inhibitors with higher bioavailability, broader spectra, and
greater efficacy.
Back to top] Is tRNA Binding or tRNA Mimicry Mandatory for
Translation Factors?
tRNA is the adaptor in the
translation process. The ribosome has three sites for tRNA, the A-, P-, and
E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site
and the mRNA on the small or 30S subunit and the peptidyl transfer site on the
large or 50S subunit. The possibility that translation release factors could
mimic tRNA has been discussed for a long time, since their function is very
similar to that of tRNA. They identify stop codons of the mRNA presented in the
decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the
peptidyl transfer site. The structures of eubacterial release factors are not
yet known, and the first example of tRNA mimicry was discovered when elongation
factor G (EF-G) was found to have a closely similar shape to a complex of
elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of
the tRNA shape is seen in ribosome recycling factor (RRF). The number of
proteins mimicking tRNA is rapidly increasing. This primarily concerns
translation factors. It is now evident that in some sense they are either tRNA
mimics, GTPases or possibly both.
Back to top] Protein Factors Mediating Selenoprotein Synthesis
The amino acid selenocysteine represents the major biological form of selenium. Both the synthesis of selenocysteine and its co-translational incorporation into selenoproteins in response to an in-frame UGA codon, require a complex molecular machinery. To decode the UGA Sec codon in eubacteria, this machinery comprises the tRNASec, the specialized elongation factor SelB and the SECIS hairpin in the selenoprotein mRNAs. SelB conveys the Sec-tRNASec to the A site of the ribosome through binding to the SECIS mRNA hairpin adjacent to the UGA Sec codon. SelB is thus a bifunctional factor, carrying functional homology to elongation factor EF-Tu in its N-terminal domain and SECIS RNA binding activity via its C-terminal extension. In archaea and eukaryotes, selenocysteine incorporation exhibits a higher degree of complexity because the SECIS hairpin is localized in the 3' untranslated region of the mRNA. In the last couple of years, remarkable progress has been made toward understanding the underlying mechanism in mammals. Indeed, the discovery of the SECIS RNA binding protein SBP2, which is not a translation factor, paved the way for the subsequent isolation of mSelB/EFSec, the mammalian homolog of SelB. In contrast to the eubacterial SelB, the specialized elongation factor mSelB/EFSec the SECIS RNA binding function. The role is carried out by SBP2 that also forms a protein-protein complex with mSelB/EFSec. As a consequence, an important difference between the eubacterial and eukaryal selenoprotein synthesis machineries is that the functions of SelB are divided into two proteins in eukaryotes. Obviously, selenoprotein synthesis represents a higher degree of complexity than anticipated, and more needs to be discovered in eukaryotes. In this review, we will focus on the structural and functional aspects of the SelB and SBP2 factors in selenoprotein synthesis.