Alap R. Subramanian

The Translational Apparatus

Robin Ray Gutell MCDBiology Campus Box 347 University of Colorado Boulder, Colorado 80309-0347 USA email: rgutell@boulder.colorado.edu The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequence Methods for more than a decade. During these years our comparative methods have evolved as the number of complete 16S and 23S rRNA sequences have increased significantly, resulting in the maturation of the higher-order structure models for 16S and 23S rRNA. With over 1000 16S (and 16S-like) and 200 23S (and 23S-like) sequences at this time, we have strong comparative evidence for the vast majority of all secondary structure base pairings, and are thus quite confident of the majority of the proposed Escherichia coli 16S and 23S rRNA secondary structure. Within the past few years additional rRNA Higher-Order structure constraints have been elucidated; constraints that reveal various RNA structural forms, including lone canonical pairings, pseudoknots, non-canonical pairings, tetra loops, canonical and non-canonical pairings that together forms a parallel (vs. the usual antiparallel) stranded structural element, and suggestive evidence for coaxial stacking of adjacent helices. At this time we question what additional RNA structural constraints can be deciphered with comparative structure methods. To answer such questions, the rRNA sequence collection will need to continue to grow in both number and diversity, and our comparative structure algorithms need to evolve to a more sophisticated level. In an effort to establish the limits for structural similarity, we need to address how different two higher-order structures can be and still be considered analogous. Introductory Statements Since the flrstcomplete 16S (Brosius et al. 1978) and 23S (Brosius et al. 1980) rRNA sequences were determined, comparative analyds of these molecules has progressed in a variety of ways. Maybe foremost for the majority (especially for this audience) is the resulting higher-order structures, which ribosome-ologists utilize to map andlor design their experiments onto. While there is a wealth of information that can and should be elucidated from the sequences that make up the 16S and 23S rRNA datasets, this article will focus on the most obvious and probably experimentally meaningful structural features, namely secondary structure helices, tertiary interactions, and a few interesting examples of other comparatively derived structural constraints. And since much has already been written on comparatively derived rRNA structure [and most recently for an upcoming book on ribosomal RNA (Gutell et al. 1993), this article will only briefly touch on some of the emerging RNA structural features and new The Translational Apparatus, Edited by K.H. Nierhaus et al., Plenum Press, New York, 1993 477 structural possibilities that have been uncovered within the past year or so, leaving the interested reader to investigate elsewhere for a more encompassing perspective of the details of the comparatively derived rRNA structures. Comparative Structure Analysis What is the basis of this method? What might we expect to decipher? This method is rooted in the simple concept that similar or analogous three-dimensional structure can be composed of different primary structures, or in other words many different primary structures can fold into the same isomorphic 3-dimensional structure. Thus natural selection can maintain and act on the higher-order structures of RNA while the primary structure is free to change, although constrained in its divergence. The ribosomal RNA is an ideal molecule to apply such methodology to due to its structural and functional role in the ribosome, and the ribosomes position in protein synthesis and the evolution of the cell (Woese 1980). Underlying this method are a number of key questions that cannot be answered a priori. How much and what types of variance can be tolerated in higher-order structure before these structures are not considered isomorphic? How much overall similarity should we expect to fmd for any RNA molecule? How much overall variance should we expect to find for any RNA molecule (i.e., tRNA: type I vs. type II)? To what extent can these methods identify general folding patterns and to what extent can these methods identify and distinguish subtle and detailed RNA structure (i.e., elucidate the generalized three-dimensional structure for tRNAs; elucidate the detailed features recognized by each of the aminoacyl synthetases for their cognate tRNA)? Will all RNA structural motifs be identified with such methods, or will only a subset of these structural elements be amenable to such methods, i.e., should we expect secondary and all tertiary interactions to be equally decipherable? And lastly, should we anticipate the same overall and/or detailed structural (and even biological) constraints within phylogenetic ally related vs. distant structures? A quick glimpse at the progression of our rRNA structure models Although these questions are not (yet) answerable, the comparative analysis of the rRNAs (and all RNAs for that matter) has advanced in stages, in part so the results from each stage with their underlying assumptions can be evalulated before moving on to the next stage, and in part due to the significant increase in the number of sequences, development of the underlying correlation analysis algorithms, and the fact that we believe there is more structural detail to be found at the completion of each stage. This analysis started with basic assumptions that were congruent with principles elucidated with experimental methods. Initially the comparative structure searched for the helices that compose the overall secondary structure [For 16S rRNA: (Woese et al. 1980, Stiegler et al. 1980, Zwieb et al. 1981), and 23S rRNA: (Nolier et al. 1981, Glotz et al. 1981, and Branlant et al. 1981) rRNAs]. These methods specifically searched for canonical base pairings (ie. A-U and G-C) arranged contiguously and in an antiparallel orientation. These structures were tested and evaluated with each new rRNA sequence, resulting in numerous refmements in the secondary structures [Many references not noted here]. The specific search for helical elements gave way to a more generalized, non-structure based method. This method (Gutell et al. 1985) transformed the pattern of nucleotides at each column in the sequence alignment to a number pattern, which was based on the pattern of conservation and variance at every position in the molecule. Similar number patterns were subsequently grouped and analyzed, resulting in refmements in the secondary structure, and several proposed tertiary interactions (Gutell et al. 1985, 1986). Equally significant this simple algorithm uncovered a few basic principles of RNA structure, namely canonical pairings, and contiguous and antiparallel arrangement of such pairings [It should be noted that this method searched for columns (nucleotide positions) with similar patterns of variation or covariance, regardless of the nucleotide and pairing types. It so happened that the underlying pairs

Primary structures of two homologous ribosome-associated DNA-binding proteins of Escherichia coli.

The proteins NSl and NS2 (NS standing for native subunit) bind specifically to the native 30 S subunit of Escherichia colz’ ribosomes [l] . The two proteins exist in solution as tetramers and have a monomer mol. wt -9500. The proteins show high similarity in amino acid composition, and they are encoded in separate genes on the E. coli chromosome [l] . Electrophoretic and immunological analyses demonstrated that the DNA-binding protein HU [2] as well as the DNA-binding protein HD [3] correspond to mixtures of NSl and NS2. The proteins belong to one of the four classes of DNA-binding proteins with regard to their molecular weights. It is highly plausible that NSl and NS2 are two individual forms of a dimorphic DNA-binding protein which specifically binds to native 30 S subunits of the ribosome. In this paper we present the complete amino acid sequences of the two proteins and show the structural relationship between them.

Molecular genetics of chloroplast ribosomal proteins.

Chloroplasts contain a complete translational apparatus which, in land plants, synthesizes the 80 or so polypeptides encoded by the organelles own small genome. Recent molecular genetic studies have revealed much about the chloroplast ribosomal proteins (RPs). Some of these proteins are encoded by the chloroplast genome and others by the nuclear genome. Many of these genes have now been cloned and characterized, including some that have no prokaryotic homologues.

Structure and functions of the largest Escherichia coli ribosomal protein

Abstract Prokaryotic ribosomes contain over fifty proteins as integral parts of their architecture, but as yet there is little precise understanding of the specific functions of individual r -proteins. In this review the special structural features and functional properties of S1 (the largest of the E. coli r -proteins), are described and a plausible model for its specific function is outlined.

The major ribosome binding site of Escherichia coli ribosomal protein S1 is located in its N-terminal segment

Abstract We have cleaved protein S1, which is the largest and the most elongated protein of the Escherichia coli ribosome, using cyanogen bromide and isolated two fragments that retain the functional domains of the intact molecule. The fragments (denoted S1-F2a and S1-F2b) showed molecular weights of 24,000 and 22,500 by dodecyl sulphate/polyacrylamide gel electrophoresis. Fragment F2a is shown to be the N-terminal segment containing about 32% of the peptide chain length of S1. Fragment F2b is derived from another (probably C-terminal) region of S1. Fragment F2a binds to 30 S ribosomal subunits with a strength and specificity comparable to the binding of intact S1. It also binds to matrix-bound poly(U) but the binding is salt-sensitive, unlike the binding of intact S1. Fragment F2b binds only very weakly to poly(U) and does not bind to 30 S subunits. These results are discussed with respect to the ribosome binding domain(s) of protein S1 and the possible interdependence of the multiple functional domains in this large protein.

Functional domains of Escherichia coli Ribosomal Protein S1. Formation and characterization of a fragment with ribosome-binding properties.

Treatment of Escherichia coli ribosomal protein S1 with TPCK-treated trypsin under mild conditions (0 °C, 1 to 2 μig trypsin/mg S1 protein) results in the production of a high molecular weight fragment in yields of up to 80% within a few minutes. The fragment is relatively resistant to further degradation. We have isolated the fragment in pure form for structural and functional characterization. The fragment (denoted S1-F1) has a molecular weight of 48,500 as shown by sodium dodecyl sulphate gel electrophoresis, and therefore it contains approximately 60% of the amino acid residues of S1. The N-terminal sequence of the fragment is different from that of intact S1. The fragment binds to the 30 S ribosomal subunit and to polyuridylic acid in approximately the same manner as intact S1, indicating that the active centres of S1 concerned with these two characteristic binding properties are localized within the fragment. In spite of the above properties, the fragment was completely unable to support protein synthesis. The significance of these results in relation to the structure and function of S1 is discussed.

Relaxation time, interthiol distance, and mechanism of action of ribosomal protein S1

The two sulfhydryl groups of ribosomal protein S1 from Escherichia coli have been labeled with fluorescent maleimides and the distance between them has been determined by nonradiative energy transfer. This distance was found to be approximately 27 A for both free S1 and S1 bound to 30 S subunits. This value probably represents an upper limit. The position of the fluorescence emission maximum indicates that both sulfhydryl groups are in a relatively hydrophobic environment. When poly(U) is added to labeled S1, either free or in 30 S subunits, the emission maximum shifts to the red by about 3 nm but without a detectable change in the interthiol distance. S1 labeled at one or both of its sulfhydryl groups retains most of its ability to enhance poly(U)-directed polyphenylalanine synthesis. About the same concentration of poly(U) is required to give the maximum shift in fluorescence as is required to give maximum polyphenylalanine synthesis, indicating that S1 binds poly(U) during translation. The peptide initiation inhibitor aurintricarboxylic acid almost completely quenches the fluorescence from either labeled sulfhydryl groups in S1 bound to ribosomes or free in solution. This quenching probably is due to energy transfer from the labeled sulfhydryls to bound aurintricarboxylic acid. Fluorescence anisotropy measurements indicated that the C-terminal domain of S1 is relatively rigid, but retains some independent movement when attached to ribosomes. The overall data are consistent with a model in which a region near the two sulfhydryl groups in the elongated C-terminal domain functions to sequester and bind mRNA to the ribosome during peptide synthesis.

Co-transcription pattern of an introgressed operon in the maize chloroplast genome comprising four ATP synthase subunit genes and the ribosomal rps2

Several examples of the introduction of a gene from one gene complex into another (introgression) are found when chloroplast RP gene clusters are compared to those in Escherichia coli or cyanobacteria. Here we describe the transcript pattern of one such cluster from maize (Zea mays) that includes the genes for 4 subunits of the thylakoid ATP synthase (atpI, H, F, A) and the rps2 gene. Twelve transcript species covering the size range from 7 000 to 800 nt were identified in RNA isolated from dark-grown and greening maize seedlings, and several of them were characterized by reverse transcription analysis. A major species of 6 200 nt, with its 5′ end at 181 nt upstream of the initiating ATG of rps2, contained the transcripts of all the 5 genes. Two further sets of transcripts having their 5′ ends ca. 120 and 50 nt upstream of the initiation codons of the atpI and atpH genes were also identified. Thus, this plastid gene cluster in maize is functionally organized as an operon with additional regulatory features to allow for increased accumulation of mRNAs for the thylakoid components.

Separation of two forms of IF-3 in Escherichia coli by two-dimensional gel electrophoresis

Among the three initiation factors which are required by ~sc~e~~c~~~ cofi ribosomes for translation of natural mRNA, IF-3 has generally been considered to play a key role. It is required to maintain a pool of free 30 S subunits [ 1,2] which normally initiate protein biosynthesis (reviewed in [3] ). In vitro IF-3 stimulates the translation of natural and synthetic messengers [3,4]. The protein has been located in the ~ei~lborhood of 16 S RNA [5] and of several ribosomal proteins that are involved in the initiation step [6] . Earlier studies also implicated a role for it in transcription [7]. Previous reports IS--lo] suggested that several forms of IF-3 may exist in E. coli, and two distinct IF-3 species were purified by Lee-Huang and Ochoa [ 11,121. The two species differed from each other in mRNA discrimination, molecular weight, isoelectric point and chromatographic behavior [ 121. Other workers could isolate only a single species of IF-3 from normal or T4 phage-infected E. COB ]4,13] . Recently we have been studying by two-dimensional gel electrophoresis the proteins specifically associated with native 30 S ribosomal particles. About 15% of the total ribosomes in E. coli extracts (in 0.01 M Mg’+) exist as native subunits. The native 30 S subunits have earlier been shown to contain initiation [ 141 and dissociation factor ]15] activities and our experiments (unpublished) show additional proteins, present stoichiometrically. During our study we obtained purified factor preparations from several laboratories for identification in two-dimensional gel systems [ 16,173 . When these preparations were analyzed, it became clear

Cloning and characterization of the genes for ribosomal proteins L10 and L12 from Synechocystis Sp. PCC 6803 : comparison of gene clustering pattern and protein sequence homology between cyanobacteria and chloroplasts

The endosymbiont theory proposes that chloroplasts have originated from ancestral cyanobacteria through a process of engulfment and subsequent symbiotic adaptation. The molecular data for testing this theory have mainly been the nucleotide sequence of rRNAs and of photosystem component genes. In order to provide additional data in this area, we have isolated genomic clones of Synechocystis DNA containing the ribosomal protein gene cluster rplJL. The nucleotide sequence of this cluster and flanking regions was determined and the derived amino acid sequences were compared to the available homologous sequences from other eubacteria and chloroplasts. In Escherichia coli these two genes are part of a larger cluster, i.e., rplKAJL-rpoBC. In Synechocystis, the genes for the RNA polymerase subunit (rpoBC) are shown to be widely separated from the r-protein genes. The Synechocystis gene arrangement is similar to that in the chloroplast system, where the rpoBC1C2 and rplKAJL clusters are separated and located in two cell compartments, the chloroplast and the nucleus, respectively.