Knud H. Nierhaus

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl‐tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo‐ electron microscopy (EM) study of an Escherichia coli 70S ribosome‐bound ternary complex stalled with an antibiotic, kirromycin. In the cryo‐EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon–anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase‐associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Ribosomal Protection Proteins and Their Mechanism of Tetracycline Resistance

Ribosomal protection represents an important tactic for promoting tetracycline resistance in both gram-positive and -negative species. Tet(O) and Tet(M) are the best studied of these determinants and were originally isolated from Campylobacter jejuni and Streptococcus spp., respectively, although both are widely distributed (10). These are the only two ribosomal protection proteins (RPPs) that have been studied in detail, and therefore, they have been dealt with extensively in this review. It is assumed, however, that the other members of this class of RPPs [Tet(S), Tet(T), Tet(Q), TetB(P), Tet(W), and OtrA] function through similar mechanisms. The distribution of these determinants in the eubacteria has been extensively reviewed by Chopra and Roberts (10) and more recent information can also be found at http://faculty.washington.edu/marilynr/. Although this review focuses primarily on RPPs, it should be noted that a great variety of tetracycline resistance mechanisms exist (for a review, see reference 10). These determinants include (i) the efflux-based mechanisms found in gram-positive and gram-negative bacteria (10), (ii) the enzymatic degradation of tetracyclines found in Bacteroides (46), (iii) the rRNA mutations found in Propionibacterium acnes and Helicobacter pylori (19, 40, 55), and (iv) a host of undetermined mechanisms which bear little resemblance to the well-documented determinants mentioned above (10). In this review, we will survey recent advances in the study of the ribosome, tetracycline, and the RPPs that further the understanding of RPP activity. Earlier work dealing with Tet(M) and Tet(O) as well as the other RPPs has been reviewed previously (51, 52).

Dissection of the Mechanism for the Stringent Factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.

Direct Visualization of A-, P-, and E-Site Transfer RNAs in the Escherichia coli Ribosome

Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond formation on the ribosome.

The assembly of prokaryotic ribosomes

The targets of in vivo studies of the ribosomal assembly process are mainly the events of rRNA processing, whereas in vitro studies (total reconstitution) focus on principles of the assembly process such as assembly-initiation proteins, rate-limiting steps and a detailed sequence of assembly reactions (assembly map). The success of in vitro analyses is particularly remarkable in view of ionic and temperature requirements of the total reconstitution which differ significantly from the in vivo conditions. Features of the in vivo assembly are surveyed, however, the focal point is a description of experimental strategies and results concerning the in vitro assembly of ribosomes.

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome.

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).

[19] Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy

Publisher Summary This chapter discusses the isolation of the ribosomes and the preparation of functional complexes and provides an overview of the possibilities for analyzing ribosomal complexes. It summarizes and discusses the results of recent cryoelectron microscopy studies that reflect the effect of buffer conditions. Studies have established that the ribosome has three transfer RNA (tRNA) binding sites, but 3-D cryo-electron microscopy (EM) has revealed five different tRNA positions on the ribosome, classified as A, P, P/E, E, and E2. The occupancy of some of these positions strongly depends on the buffer conditions used and the charge state of the tRNA. In the presence of the polyamine buffer, mimicking the in vivo conditions, only occupancy of A, P, and E sites are observed in complexes of the initiating and elongating ribosomes. The procedure described in the chapter for the small-scale isolation of tightly coupled ribosomes yields highly active and intact ribosomes, an important prerequisite for the preparation of functional complexes. The chapter describes the isolation of ribosomal subunits that can be used to prepare reassociated ribosomes. Reassociated ribosomes show a more efficient tRNA binding as compared to tightly coupled ribosomes, because the saturation of tRNA binding is reached at molar ratios slightly above stoichiometric ones. This can be attributed to at least two factors: (1) a selective pressure for active particles in the reassociation step and (2) the loss of residual amounts of tRNAs and of mitochondrial RNA (mRNA) fragments.

Evidence that the G2661 region of 23S rRNA is located at the ribosomal binding sites of both elongation factors.

Alpha-sarcin cleaves one phosphodiester bond of 23S rRNA within 70S ribosomes or 50S subunits derived from E. coli. The resulting fragment was isolated and sequenced. The cleavage site was identified as being after G2661 and is located within a universally conserved dodecamer. Cleavage after G2661 specifically blocked the binding of both elongation factors, i.e. that of the ternary complex Phe-tRNA*EF-Tu*GMPPNP and of EF-G*GMPPNP, whereas all elongation-factor independent functions of the ribosome, such as association of the ribosomal subunits, tRNA binding to A and P sites, the accuracy of tRNA selection at both sites, the peptidyl transferase activity, and the EF-G independent, spontaneous translocation, were not affected at all. Control experiments with wheat germ ribosomes yielded an equivalent inhibition pattern. The data suggest that the universally conserved dodecamer containing the cleavage site G2661 is located at the presumably overlapping region of the binding sites of both elongation factors.

Ribosomal Proteins in the Spotlight

ABSTRACT The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.

The Weird and Wonderful World of Bacterial Ribosome Regulation

ABSTRACT In every organism, translation of the genetic information into functional proteins is performed on the ribosome. In Escherichia coli up to 40% of the cells total energy turnover is channelled toward the ribosome and protein synthesis. Thus, elaborate networks of translation regulation pathways have evolved to modulate gene expression in response to growth rate and external factors, ranging from nutrient deprivation, to chemical (pH, ionic strength) and physical (temperature) fluctuations. Since the fundamental players involved in regulation of the different phases of translation have already been extensively reviewed elsewhere, this review focuses on lesser known and characterized factors that regulate the ribosome, ranging from processing, modification and assembly factors, unusual initiation and elongation factors, to a variety of stress response proteins.