The distance between S1, S21, and the 3′ end of 16S RNA in 30S ribosomal subunits

Abstract

The apparent distances between probes covalently attached to the cysteine thiols of S1 or S21 and the 3' end of 16s RNA in Escherichia coli 30s ribosomal subunits were determined by non-radiative energy transfer to be: S21-16s RNA, 5.1 nm; S22 -S1, 6.9 nm; S1-16s RNA, 6.8 nm. Binding ofpoly(uridy1ic acid) to 30s subunits causes the apparent distance$ between S1 and 16s RNA or S21 and 16s RNA to increase by more than 1.2 nm and 0.5 nm, respectively, but has little or no effect on the S1-S21 distance. Binding of 50s subunits causes an apparent increase in the S21-16s RNA and S21-S1 distances by 1 .O nm and 0.8 nm, respectively, but has little or no effect on the S1-16s-RNA distance. S1 with 557 amino acid residues [I] is the largest protein of the Escherichia coliribosome. It is highly elongated [2 -61 with a length similar to the maximum dimension of the 30s subunit to which it is bound by its trypsin-sensitive N-terminal region. This domain extends to residue 171 171. The C-terminal, trypsin-resistant segment contains an RNA binding site and two cysteine residues at positions 292 and 349 [I, 6,7]. Its thiol groups can be labeled with fluorescent probes without inactivating the protein for poly(U) translation [8]. The exact function of S1 in QP replicase [9] or in peptide synthesis is not known; however, it has been proposed that mRNA is sequestered for translation by binding, to the elongated C- RNA [13] and an octadeoxynucleotide which is complementary to the 3' end of 16s RNA [14]. It has been proposed that it functions to hold the 3' end of 16s RNA in a conformation in which it can bind to the Shine-Dalgarno sequence of natural mRNA [13]. Immunoelectron microscopy studies of S3 [15,16], S21 [15,17], and the 3'end of 16s RNA [18-201 have placed all of these components in the same general area of the 30s subunit near the constriction that separates the 'head' from the 'body'. There have been several additional studies that place S21 and S1 near each other in the vicinity of the 3' end of 16s RNA. Both proteins have been cross-linked to the 3' end of 16s RNA [21] as well as to IF-3Abbreviations. CPM, 3-(4-maleimidylphenyI)-4-methyl-7-(diethylamino)coumarin; FTS, fluorescein thiosemiicarbazide; FM, fluorescein 5-maleimide; CPM-S21, S21 labeled at,its sulfhydryl group with CPM; 16s 16s RNA labeled at 'its 3' end with FTS; FM-S1, S1 labeled at its sulfhydryl groups with FM; poly(U), polyuridylic acid ; Hepes, 4-(2-hydroxymethyl)-l-piperazineethanesulfonic acid; 30S( -Sl), 30S( -S21), and 30S(-S21,-Sl), '30s subunits lacking the proteins indicated in parentheses ; Mg(OAe)2, magnesium acetate. ~-[22] and to protein S18 [23]. Poly(4-thiouridylic acid) bound to 70s ribosomes was shown to react upon photoactivation with both S21 and S1 as well as with S18 [24].Here we report the distances involved in the triangle formed by probes on S21, S1 and the 3' end of 16s RNA in ribosomal subunits, as determined from singlet-singlet fluorescence energy transfer. We also report the effect of poly(U) and 50s subunits on these di...

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

“…Previous workers have reconstituted 30S or 50S subunits in which a single protein has been modified, either to test functional consequences (Kahan et al, 1974;Shimizu & Craven, 1976;Hernandez et al, 1977;Baxter & Zahid, 1978;Auron et al, 1978;Baxter 3 A special case where such a function can be tested occurs when a new function is conferred by virtue of the photoaffmity labeling process-see, for example, Hsiung et al (1974). et al, 1980) or to introduce a probe for electron microscopy or fluorescence energy transfer studies (Stoffler-Meilicke et al, 1983Maasen et al, 1984;Odom et al, 1984), but this earlier work involved non-site-specific modification of ribosomal proteins to high stoichiometries with group-specific reagents. The advantage of our approach is that because modification proceeds from a specific site with a functionally important ligand, there is a much clearer rationale for interpreting changes in function of the reconstituted particle as reflecting specific modification of a functional site.…”

Structure and function of ribosomal RNA

“…In addition, the distances between the 3'-ends of 5S, 16S and 23S RNA in 70S ribosomes are known from energy transfer measurements with fluorescent probes (Odom et al, 1980), and the distances between the 3'-end of 16S RNA and probes attached to proteins SI and S21 have similarly been determined in various functional states (e.g. Odom et al, 1984a). In this context the two highly specific cross-links to tRNA already discussed (see section on inter-RNA interaction) represent in effect a 'distance measurement' between residues 1400 in 16S RNA and residues 2584/5 in the 23S RNA (cf.…”

Ensemble and single-molecule FRET studies of protein synthesis

“…The Hardesty’s group, which pioneered the use of FRET for probing of the ribosomal structure, labeled the 3’ end of 16S rRNA through periodate oxidation and then reconstituted the small (30S) subunit from fluorescently-labeled 16S rRNA and a mixture of 30S proteins (TP30) [69, 70]. More recently, a different strategy for labeling rRNA was developed: fluorescently-labeled oligonucleotides were hybridized with 22–23 nucleotide-long extensions of 16S and 23 rRNA, which were introduced via mutagenesis to replace the loops in surface-exposed rRNA helices [71].…”

“…Most commonly, a ribosomal protein conjugated to a fluorophore is introduced into ribosomes, which lack the same protein due to either chemical or genetic manipulations. Proteins bS21 and bL12 can be selectively depleted from E. coli ribosomes by NH 4 Cl and NH 4 Cl/ethanol treatments, respectively [69, 74]. In addition, there are a number of ribosomal proteins that are not essential for cell viability and can therefore be deleted from the bacterial genome one or two at a time.…”