The prevailing "plug-in-the-bottle" model suggests that macrolide antibiotics inhibit translation by binding inside the ribosome tunnel and indiscriminately arresting the elongation of every nascent polypeptide after the synthesis of six to eight amino acids. To test this model, we performed a genome-wide analysis of translation in azithromycin-treated Staphylococcus aureus. In contrast to earlier predictions, we found that the macrolide does not preferentially induce ribosome stalling near the 5′ end of mRNAs, but rather acts at specific stalling sites that are scattered throughout the entire coding region. These sites are highly enriched in prolines and charged residues and are strikingly similar to other ligandindependent ribosome stalling motifs. Interestingly, the addition of structurally related macrolides had dramatically different effects on stalling efficiency. Our data suggest that ribosome stalling can occur at a surprisingly large number of low-complexity motifs in a fashion that depends only on a few arrest-inducing residues and the presence of a small molecule inducer.antibiotic | ribosome stalling | Staphylococcus aureus

Many naturally occurring RNA structures contain single mismatches. However, the algorithms currently used to predict RNA structure from sequence rely on a minimal set of data for single mismatches, most of which occur rather infrequently in nature. As a result, several approximations and assumptions are used to predict the stability of RNA duplexes containing the most common single mismatches. Therefore, the relative frequency of single mismatches was determined by compiling and searching a database of 955 RNA secondary structures. Thermodynamic parameters for duplex formation, derived from optical melting experiments, are reported for 28 oligoribonucleotides containing frequently occurring single mismatches. These data were then combined with previous data to construct a dataset of 64 single mismatches, including the 30 most common in the database. Because of this increase in experimental thermodynamic parameters for single mismatches that occur frequently in nature, more accurate free energy calculations have resulted. To improve the prediction of the thermodynamic parameters for duplexes containing single mismatches that have not been experimentally measured, single mismatch-specific nearest neighbor parameters were derived. The free energy of an RNA duplex containing a single mismatch that has not been thermodynamically characterized can be calculated by: DeltaG degrees 37,single mismatch = DeltaG degrees 37,mismatch nt + DeltaG degrees 37,mismatch-NN interaction + DeltaG degrees 37,AU/GU. Here, DeltaG degrees 37,mismatch is -0.4, -2.1, and -0.3 kcal/mol for A.G, G.G, and U.U mismatches, respectively; DeltaG degrees 37,mismatch-NN interaction is 0.7, -0.5, 0.4, -0.4, and -1.0 kcal/mol for 5'YRR3'/3'RRY5', 5'RYY3'/3'YYR5', 5'YYR3'/3'RYY5', 5'YRY3'/3'RYR5', and 5'RRY3'/3'YYR5' mismatch-nearest neighbor combinations, respectively, when A and G are categorized as purines (R) and C and U are categorized as pyrimidines (Y); and DeltaG degrees 37,AU/GU is a penalty of 1.2 kcal/mol for replacing a G-C base pair with either an A-U or G-U base pair. Similar predictive models were also derived for DeltaH degrees single mismatch and DeltaS degrees single mismatch. These new predictive models, in conjunction with the reported thermodynamics for frequently occurring single mismatches, should allow for more accurate calculations of the free energy of RNA duplexes containing single mismatches and, furthermore, allow for improved prediction of secondary structure from sequence.

Although tetraloops are one of the most frequently occurring secondary structure motifs in RNA, less than one-third of the 30 most frequently occurring RNA tetraloops have been thermodynamically characterized. Therefore, 24 stem-loop sequences containing common tetraloops were optically melted, and the thermodynamic parameters DH°, DS°, DG°3 7, and T M for each stem-loop were determined. These new experimental values, on average, are 0.7 kcal/mol different from the values predicted for these tetraloops using the model proposed by Vecenie CJ, Morrow CV, Zyra A, Serra MJ. 2006. Biochemistry 45: 1400-1407. The data for the 24 tetraloops reported here were then combined with the data for 28 tetraloops that were published previously. A new model, independent of terminal mismatch data, was derived to predict the free energy contribution of previously unmeasured tetraloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.4 kcal/mol. This new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing tetraloops and, furthermore, should allow for improved prediction of secondary structure from sequence. It was also shown that tetraloops within the sequence 59-GCCNNNNGGC-39 are, on average, 0.6 kcal/mol more stable than the same tetraloop within the sequence 59-GGCNNNNGCC-39. More systemic studies are required to determine the full extent of non-nearest-neighbor effects on tetraloop stability.

RNA secondary structure is important for designing therapeutics, understanding protein–RNA binding and predicting tertiary structure of RNA. Several databases and downloadable programs exist that specialize in the three-dimensional (3D) structure of RNA, but none focus specifically on secondary structural motifs such as internal, bulge and hairpin loops. The RNA Characterization of Secondary Structure Motifs (RNA CoSSMos) database is a freely accessible and searchable online database and website of 3D characteristics of secondary structure motifs. To create the RNA CoSSMos database, 2156 Protein Data Bank (PDB) files were searched for internal, bulge and hairpin loops, and each loop's structural information, including sugar pucker, glycosidic linkage, hydrogen bonding patterns and stacking interactions, was included in the database. False positives were defined, identified and reclassified or omitted from the database to ensure the most accurate results possible. Users can search via general PDB information, experimental parameters, sequence and specific motif and by specific structural parameters in the subquery page after the initial search. Returned results for each search can be viewed individually or a complete set can be downloaded into a spreadsheet to allow for easy comparison. The RNA CoSSMos database is automatically updated weekly and is available at http://cossmos.slu.edu.

RNA is known to be involved in several cellular processes; however, it is only active when it is folded into its correct 3D conformation. The folding, bending and twisting of an RNA molecule is dependent upon the multitude of canonical and non-canonical secondary structure motifs. These motifs contribute to the structural complexity of RNA but also serve important integral biological functions, such as serving as recognition and binding sites for other biomolecules or small ligands. One of the most prevalent types of RNA secondary structure motifs are single mismatches, which occur when two canonical pairs are separated by a single non-canonical pair. To determine sequence–structure relationships and to identify structural patterns, we have systematically located, annotated and compared all available occurrences of the 30 most frequently occurring single mismatch-nearest neighbor sequence combinations found in experimentally determined 3D structures of RNA-containing molecules deposited into the Protein Data Bank. Hydrogen bonding, stacking and interaction of nucleotide edges for the mismatched and nearest neighbor base pairs are described and compared, allowing for the identification of several structural patterns. Such a database and comparison will allow researchers to gain insight into the structural features of unstudied sequences and to quickly look-up studied sequences.

Due to their prevalence and roles in biological systems, single mismatches adjacent to G-U pairs are important RNA structural elements. Since there are only limited experimental values for the stability of single mismatches adjacent to G-U pairs, current algorithms using free energy minimization to predict RNA secondary structure from sequence assign predicted thermodynamic values to these types of single mismatches. Here, thermodynamic data are reported for frequently occurring single mismatches adjacent to at least one G-U pair. This experimental data can be used in place of predicted thermodynamic values in algorithms that predict secondary structure from sequence using free energy minimization. When predicting the thermodynamic contributions of previously unmeasured single mismatches, most algorithms apply the same thermodynamic penalty for an A-U pair adjacent to a single mismatch and a G-U pair adjacent to a single mismatch. A recent study, however, suggests that the penalty for a G-U pair adjacent to a tandem mismatch should be 1.2 +/- 0.1 kcal/mol, and the penalty for an A-U pair adjacent to a tandem mismatch should be 0.5 +/- 0.2 kcal/mol [Christiansen, M. E. and Znosko, B. M. (2008) Biochemistry 47, 4329-4336]. Therefore, the data reported here are combined with the existing thermodynamic dataset of single mismatches, and nearest neighbor parameters are derived for an A-U pair adjacent to a single mismatch (1.1 +/- 0.1 kcal/mol) and a G-U pair adjacent to a single mismatch (1.4 +/- 0.1 kcal/mol).

Many naturally occurring RNA structures contain single mismatches, many of which occur near the ends of helices. However, previous thermodynamic studies have focused their efforts on thermodynamically characterizing centrally placed single mismatches. Additionally, algorithms currently used to predict secondary structure from sequence are based on two assumptions to predict stability of RNA duplexes containing this motif. It has been assumed that the thermodynamic contribution of small RNA motifs is independent of both its position in the duplex and identity of the non-nearest neighbors. Thermodynamically characterizing single mismatches three nucleotides from both the 3′ and 5′ ends (i.e., off-center) of an RNA duplex and comparing these results to those of the same single mismatch-nearest neighbor combination centrally located has allowed for the investigation of these effects. The thermodynamic contribution of 13 single mismatch-nearest neighbor combinations are reported but only 9 combinations are studied at all three duplex positions and are used to determine trends and patterns. In general, the 5′ and 3′ shifted single mismatches are relatively similar, on average, and more favorable in free energy than centrally placed single mismatches. However, close examination and comparison shows there are several associated idiosyncrasies with these identified general trends. These peculiarities may be due, in part, to the identities of the single mismatch, the nearest neighbors, and the non-nearest neighbors, along with the effects of single mismatch position in the duplex. The prediction algorithm recently proposed by Davis and Znosko (Biochemistry 47, 10178–10187) is used to predict the thermodynamic parameters of single mismatch contribution and is compared to the measured values presented here. This comparison suggests the proposed model is a good approximation but could be improved by the addition of parameters which account for positional and/or non-nearest neighbor effects. However, more data is required to better understand these effects and to accurately account for them.

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