Unfolding of mrna Secondary Structure by the Bacterial Translation Initiation Complex

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1 Molecular Cell 22, , April 7, 2006 ª2006 Elsevier Inc. DOI /j.molcel Unfolding of mrna Secondary Structure by the Bacterial Translation Initiation Complex Sean M. Studer 1 and Simpson Joseph 1, * 1 Department of Chemistry and Biochemistry University of California, San Diego 9500 Gilman Drive La Jolla, California Summary Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mrna. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mrnas that form secondary structures with various stabilities and contain Shine- Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mrna structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator trna, the association kinetics of structured mrnas showed two distinct phases. The second phase was found to be important for unfolding structured mrnas to form a stable 30S initiation complex. We show that unfolding of structured mrnas requires a SD sequence, the start codon, fmet-trna fmet, and the GTP bound form of initiation factor 2 bound to the 30S subunit. Introduction A widespread mechanism for translational control of gene expression is through modulation of the secondary structure at the translational initiation region (TIR) of mrna (de Smit, 1998). Specialized structures in the TIR cause translation of certain mrnas to respond to the level of a protein in the cell (Draper, 1993; Nomura et al., 1984), to the concentration of a metabolite (for example, the recently discovered riboswitches and ribozymes) (Mandal and Breaker, 2004), or to changes in the temperature (Hoe and Goguen, 1993; Johansson et al., 2002). Thus, exquisite control of gene expression can be achieved at the translational level by mrna secondary structure. A simplified view of translation initiation in bacteria involves the 30S ribosomal small subunit interacting with an mrna, three initiation factors (IF1, 2, and 3), and initiator trna (fmet-trna fmet ) (Boelens and Gualerzi, 2002). The process of initiation becomes more complicated when secondary structure is present in the TIR of the mrna. The complication arises because the ribosome must overcome the thermodynamic barrier of mrna secondary structure to form a stable mrna30s complex. Two models have been proposed to explain this dilemma. One model involves the 30S subunit directly binding to a folded mrna while it briefly exists in a transient unfolded state. However, this model is *Correspondence: sjoseph@chem.ucsd.edu not feasible due to the kinetics of mrna secondary structure formation (de Smit and van Duin, 2003). An alternative model utilizes the standby site. In this model, the 30S subunit likely binds to single-stranded regions (standby sites) of the mrna and then slides into place when the TIR unfolds (de Smit and van Duin, 2003; Draper, 1993). The SD sequence, present in the TIR, then base pairs with the anti-sd sequence (ASD) present at the 3 0 end of the 16S rrna. The SD-ASD interaction stabilizes the mrna on the 30S initiation complex. The mechanism of recruitment of structured mrnas by the 30Sinitiation factor complex is not clear. It is plausible that initiation factors (IFs) and fmet-trna fmet may affect the dynamics of mrna binding to the 30S subunit. Therefore, we developed several fluorescence-based assays to systematically study both the binding of model mrnas to the 30S subunit and the role the three bacterial IFs play in this process. A steadystate assay was used to determine the equilibrium binding constants (K D ) of the different mrnas for the 30S subunit. A transient-state assay was used to quantitatively examine the kinetics of structured and unstructured mrnas interaction with the 30S initiation complex. In addition, we developed a new assay based on fluorescence resonance energy transfer (FRET) to directly monitor the disruption of an mrna hairpin by the bacterial translation initiation complex. Our results showed that the 30S initiation complex bound to structured mrnas in two steps: an initial binding step to a single-stranded region of the mrna, followed by a distinct mrna restructuring step that required the SD sequence, the start codon, fmet-trna fmet, and IF2GTP. Results Design of Model mrnas We synthesized several model mrnas to study how mrna secondary structure and SD sequence affected binding of mrna to the 30S initiation complex. The model mrnas were designed using mfold software (Zuker, 2003) and were predicted to form secondary structures of defined stability (Figure 1A). The mrnas were named as follows: the number indicates the number of nucleotides that make up the SD sequence, and the letter indicates the relative thermal stability of the mrna. For example, all mrnas containing the number 6 have a 6 nt long SD sequence. The letter, which follows the number, indicates the thermal stability of the mrna, relative to the mrnas that contain the same length SD sequence. For example, mrna6a is predicted to be more stable than mrna6b, and mrna6c is predicted to be less stable than mrna6b, and these mrnas all contain a 6 nt SD sequence. The mrnas were analyzed by thermal denaturation studies (T m ) and native gel electrophoresis in order to determine the presence and stability of the mrna s secondary structure. The mrnas were first analyzed by thermal denaturation studies (T m ). As expected, mrna6a has the highest T m (w70ºc, DG =24.8 kcal/mol) followed by mrna6b (w49ºc, DG = 21.4 kcal/mol),

2 Molecular Cell 106 Figure 1. Sequence and Folding of the mrnas (A) Secondary structure model of the mrnas. Red and green nucleotides correspond to the SD sequence and the initiation codon, respectively. The yellow star indicates the pyrene probe at the 3 0 end of the mrna. The experimentally determined melting temperatures (T m ) of the mrnas are indicated. (B) Folding of mrnas analyzed by native gel electrophoresis. The mrnas are indicated above the lanes. mrna4a (w44ºc, DG = 20.5 kcal/mol), mrna8a (w34ºc, DG = 0.3 kcal/mol), mrna0a (w25ºc, DG = 1 kcal/mol), and mrna6c (20ºC, DG = 0.7 kcal/mol) (Figure 1). It was difficult to accurately determine the T m of mrna8b, mrna4b, and mrna0b because they lacked stable secondary structures. In order to complement the thermal denaturation studies, the mrnas were also analyzed by native gel electrophoresis. As expected, mrna0b (polyu) does not form any secondary structure and migrated the slowest while mrna6a formed a very stable hairpin structure and migrated the fastest on the gel. The other mrnas formed structures of intermediate stabilities and migrated to varying distances depending on their secondary structure. mrna8b (polya) has a run of several adenines that are known to stack (Holder and Lingrel, 1975); hence, it migrated further than the unstructured mrna0b (polyu). mrna4b was predicted to have two alternate secondary structures, and the native gel, as expected, showed two bands. Overall, there was a strong correlation between the T m of the mrna and the rate with which it migrated on the native gel. Monitoring mrna Binding to the 30S Subunit After we developed an mrna library, we wanted to examine how secondary structure and SD sequence affected the individual mrna s affinity for the 30S subunit. In order to monitor the interaction of mrnas with the 30S subunit, the fluorescent probe pyrene was covalently linked to the 3 0 terminus of the mrna. Association of mrna with the 30S subunit increased the fluorescence intensity of the pyrene probe by 3- to 4-fold. Prebinding of unlabeled mrna to the 30S subunit inhibited the increase in fluoresence intensity observed when pyrene-labeled mrna was added; this indicated that the mrnas bound to a specific site in the 30S subunit (see Figure S1A in the Supplemental Data available with this article online). Addition of pyrene-labeled mrnas to the 50S ribosomal large subunit did not result in any significant change in fluorescence intensity, also indicating that the mrnas interacted specifically with the 30S subunit (Figure S1A). Interestingly, mrna6a, which formed the most stable hairpin structure with a GNRA tetraloop, showed very little change in fluorescence intensity when mixed with the 30S subunit (Figure S1B). Thus, mrna6a served as a control demonstrating the absence of nonspecific interaction with the 30S subunit. Equilibrium binding studies were perfomed by keeping the mrna concentration fixed and changing the concentration of the 30S subunit over a wide range. These studies showed that mrnas with weaker secondary structures bound tightly to the 30S subunit (Table S1). For example, mrna6c (K D = nm) has an z238-fold higher affinity for the 30S subunit than mrna6b (K D = nm). Similarly, mrna4b (K D = nm) bound z46-fold more tightly to the 30S subunit than mrna4a (K D = nm). In addition, the stability of the SD-ASD interaction also affected the equilibrium binding constants of the mrna for the 30S subunit. mrna8a and 8b bound with such high affinities that it was impossible to accurately determine their equilibrium binding constants (mrna8a K D % and mrna8b K D % nm). When we compared other weakly structured mrnas with different SD lengths, it was found that mrna6c had a 7-fold higher affinity than mrna4b. Interestingly, mrna0a and mrna0b also bound tightly to the 30S subunit even though they lacked a SD sequence. The high affinity of these mrnas for the 30S subunit may be due to the weak secondary structures in these mrnas. Thus, mrna secondary structure and the stability of the SD- ASD interaction affected the overall equilibrium binding affinity of the mrna to the 30S subunit. The equilibrium

3 Unfolding of Structured mrnas during Translation 107 binding data also indicated that the mrnas were binding specifically to the 30S subunit. mrna Secondary Structure Inhibits Association In cells, protein expression is partially dependent on the binding kinetics of mrnas with the 30S initiation complex. Therefore, we wanted to examine the effect that the SD sequence and the mrna secondary structure had in affecting the association kinetics with the 30S subunit. The increase in fluorescence intensity, caused by the interaction of the mrna with the 30S subunit, was used to monitor the transient-state kinetics of mrna binding to the 30S subunit. First, binding of mrna to the 30S subunit was performed in the absence of fmet-trna fmet and the IFs. We observed that there was a significant correlation between the mrna association kinetics and the extent of secondary structure present in each mrna (Figure 2A and Table 1). However, there was no correlation between the length of the SD sequence and the rate constant of mrna association. mrna0 (polyu), which lacked secondary structure, had the fastest association rate constant (k on = mm 21 s 21 ) followed by mrna4b (k on = mm 21 s 21 ). Similarly, mrna6c (k on = mm 21 s 21 ), mrna0a (k on = mm 21 s 21 ), and mrna8b (k on = mm 21 s 21 ), which formed weak secondary structures, associated rapidly with the 30S subunit. In contrast, the mrnas that contained the most stable structures mrna8a (k on = mm 21 s 21 ), mrna4a (k on = mm 21 s 21 ), and mrna6b (k on = mm 21 s 21 ) all bound at a slower association rate constant than the unstructured mrnas. These data indicated that, in fact, SD length did not affect mrna association kinetics, while the presence of mrna secondary structures significantly impaired the mrna association rate. Additionally, mrna6a, which formed a stable hairpin with a GNRA tetraloop, did not bind to the 30S subunit. Based on the results with mrna6a, we reasoned that the presence of a single-stranded region (standby site) may permit mrna6a to interact with the 30S subunit. Therefore, we synthesized an extended version of mrna6a with 12 additional uridine residues at the 5 0 terminus (Figure 1A). This extended mrna, mrna6a (Ext), bound to the 30S subunit as predicted (k on = mm 21 s 21 )(Table 1, Figures S1B and S1C). This result demonstrated that a single-stranded region in a structured mrna permitted it to bind to the 30S subunit. Effect of IFs and Initiator trna on mrna Binding Two alternative pathways for mrna binding to the translation initiation complex in bacteria have been proposed (Boelens and Gualerzi, 2002). mrnas can bind either to the 30S subunit associated with the three IFs or to the 30S subunit associated with the three IFs plus fmettrna fmet in the P site. Therefore, we determined the mrna association rate constant with the 30S subunit, with the 30S subunit complexed with the IFs, and the 30S subunit complexed with the IFs plus fmet-trna fmet (Figure 2C and Table 1). Surprisingly, there were no differences in the rate constants observed for the association of mrna with either the 30S subunit (no IFs and no fmet-trna fmet ), 30S complexed with IFs (no fmettrna fmet ), or 30S complexed with IFs and fmettrna fmet. This indicated that IFs and fmet-trna fmet Table 1. Kinetic Parameters for mrna Association, k on (mm 21 s 21 ) mrna 30S 30S + IFs 30S + IFs + fmet-trna fmet 8a b a Ext b c a b a b polyu The kinetic constants represent average values from at least three different experiments, and the errors are standard uncertainties. IFs denotes all three IFs. had no effect on the kinetics of the initial interaction of the mrna with the 30S subunit. However, in the case of structured mrna6b, after the initial rapid increase in fluorescence intensity, a slower increase in fluorescence intensity was observed at longer time points (k 2 = s 21 )(Figure 2C, green trace). This only occurred in the presence of IFs and fmettrna fmet. The slow increase in fluorescence intensity was also observed when fmet-trna fmet was added to mrna6b prebound to the 30S subunit containing the three IFs (Figure 2D, green trace). Importantly, addition of deacylated trna fmet did not trigger the slow increase in fluorescence (Figure 2D, red trace), indicating that this process is relevant to the initiation pathway. Furthermore, the rate of the second phase was the same whether 2-fold or 10-fold excess fmet-trna fmet was added (data not shown), indicating that trna binding was not the rate-limiting step for the second phase. Titration experiments with a large excess of IFs (20-fold over the 30S subunit concentration) also showed similar rate constants for the second phase (data not shown), indicating that binding of IFs to the 30S subunit were not rate limiting for the second phase. Comparable results were observed with the extended mrna6a (Ext), which forms a stable secondary structure (T m = 70ºC, DG = 24.8 kcal/mol) (Figure S1C). In contrast, mrna6a was still unable to bind to the 30S subunit even in the presence of fmet-trna fmet and IFs (data not shown). We reasoned that the slow second phase may reflect the repositioning of the mrna on the 30S subunit (Canonaco et al., 1989; La Teana et al., 1995) or may be due to the disruption of mrna structure followed by productive binding to the 30S subunit. IFs and fmet-trna fmet Stabilize mrnas on the 30S Subunit In order to further analyze the interaction between the 30S subunit and the mrna, the dissociation rate constants of the mrnas were determined. The dissociation of fluorescently labeled mrna from the 30S initiation complex was monitored by the decrease in fluorescence intensity when mixed with an excess of unlabeled mrna (Figure 2E). The dissociation of all mrnas, except mrna8b, which showed little dissociation, was determined for three types of complexes: (1) mrna+30s, (2) mrna+30s+ifs, and (3) mrna+30s+ifs+fmet-trna fmet. Results showed that mrna dissociation rate constants

4 Molecular Cell 108 Figure 2. Transient-State Association and Dissociation Kinetics of mrna (A) Time course of mrna binding to the 30S subunit measured by stopped-flow analysis. (B) Association kinetics at the indicated mrna6b concentrations. The concentration of the 30S subunit was 120 nm, and the change in fluorescence was normalized to arbitrary values. (C) Time course of mrna6b binding to the different 30S complexes as indicated. (D) Change in fluorescence intensity when fmet-trna fmet (green trace) or deacylated trna fmet (red trace) are added to the 30S subunits complexed with mrna6b. (E) Dissociation of mrna6b from the 30S subunit in the absence (red trace) and presence of the IFs (green trace). (F) Dissociation of mrna6b from the 30S subunit complexed with all three IFs and fmet-trna fmet. Table 2. Kinetic Parameters for mrna Dissociation, k off (s 21 ) mrna 30S 30S + IFs 30S + IFs + fmet-trna fmet 8a a Ext b c a b a b polyu The kinetic constants represent average values from at least three different experiments, and the errors are standard uncertainties. IFs denotes all three IFs. were affected by the length of the SD sequence, the extent of mrna secondary structure, and the presence of IFs and fmet-trna fmet (Table 2). The mrna dissociation rate constants for mrna8a (k off = s 21 ) and 8b were the slowest. The dissociation rate constant of mrna8b was not determined due to the small change in signal. The mrnas that contained a 6 nt SD sequence showed a range of dissociation rate constants that correlated with their secondary structure. mrna6c exhibited the slowest rate of dissociation (k off = s 21 ), followed by mrna6b (k off = s 21 ) and mrna6a (Ext) (k off = s 21 ). The changes in the dissociation rate constants are most likely due to differences in the thermal stability of each mrna. mrna6c, which showed the slowest dissociation rate constant of the three mrnas, also contained the least stable secondary structure, as opposed to mrna6a (Ext), which contained the most extensive secondary structure and dissociated with the fastest rate constant. The other mrnas that contained either a 4 nt SD sequence or no SD sequence all dissociated at approximately the same rate. The nearly identical dissociation rates of these mrnas indicated that the 4 bp SD sequence did not significantly stabilize the mrna on the 30S subunit. Since the dissociation rate constants of the mrnas complexed with the 30S subunit or 30S + IFs were similar, we reasoned that there was no additional stabilization of these mrnas on the 30S by the three IFs. However, in the presence of IFs plus fmet-trna fmet, all of the mrnas, except mrna 0b (polyu), showed a decreased dissociation rate constant. Remarkably, the dissociation rate constant of the most structured mrnas

5 Unfolding of Structured mrnas during Translation 109 Table 3. Kinetic Parameters for mrna Dissociation, k off (s 21 )in the Presence of Various Combinations of Factors Complex mrna 6b 30S S + IFs** S + fmet-trna fmet S + IF1 + fmet-trna fmet S + IF3 + fmet-trna fmet S + IF1 + IF3 + fmet-trna fmet S + IF2 + IF3 + fmet-trna fmet S + IFs**+ fmet-trna fmet S + IF2 + fmet-trna fmet S + IF1 + IF2 + fmet-trna fmet The kinetic constants represent average values from at least three different experiments, and the errors are standard uncertainties. IFs** denotes all three IFs. (mrna6b and mrna6a [Ext]), from the 30SIFsfMettRNA fmet complex, were reduced by 100-fold and 300- fold, respectively. (Figure 2F and Figure S1E). Furthermore, mrna6c, 6b and 6a (Ext) showed less than a 2-fold difference in their dissociation rate constants from the 30SIFsfMet-tRNA fmet complex. This suggested that IFs plus fmet-trna fmet are required for the stable interaction of mrnas having secondary structure with the 30S subunit. Based on the dissociation data and the secondary structures of the mrnas, we reasoned that it was feasible for IFs and fmet-trna fmet to facilitate unfolding of structured mrnas on the 30S subunit by promoting the interaction between the SD-ASD sequences. Once SD-ASD base pairs are established, then disruption of the 6 bp SD-ASD interaction becomes rate limiting for the dissociation of mrna6a (Ext), mrna6b, and mrna6c from the 30S initiation complex. Effect of Individual Factors on the Rates of mrna Dissociation Next, factor omission experiments were performed to precisely identify which IFs are required for the slower dissociation rate of mrna6b from the 30S initiation complex. Our studies showed that IF2GTP and fmettrna fmet were the only components required to significantly slow the dissociation of the mrna from the 30S subunit (k off = s 21 ). Suprisingly, this rate was significantly slower than in the presence of all three IFs and fmet-trna fmet (k off = s 21 ). This suggested that IF1 and IF3 may play additional roles in affecting the stability of mrna in the 30S subunit. Further analysis showed that IF2 plus IF1 decreased the mrna dissociation rate constant, while IF2 plus IF3 increased the mrna dissociation rate constant (Table 3). Thus, IF1 and IF3 seemed to conteract each other to fine tune the stability of the mrna in the 30S initiation complex. Since binding of IFs is cooperative, factor omission experiments were repeated at several-fold higher concentration (5 mm final concentration) of IF1 and IF3 over the reported binding constants (Weiel and Hershey, 1981; Zucker and Hershey, 1986). Even at these higher concentrations of IFs, the dissociation rate constants remained unchanged. Therefore, we concluded that IF1 and IF3 were unable to stabilize mrna6b on the 30S subunit. Unfolding of mrna by the 30S Initiation Complex In order to directly test the hypothesis that mrna secondary structure may be disrupted on the 30S subunit by IFs and fmet-trna fmet, we synthesized mrna6b with the fluorescent probes cyanin 3 (cy3) and cyanin 5 (cy5) covalently attached to the 5 0 and 3 0 termini of the mrna, respectively (Figure 3A). mrna6b was selected for the FRET studies because the 5 0 and 3 0 ends of the mrna are close together. Thermal melting analysis showed that the cy3/cy5-labeled mrna6b formed a slightly more stable structure (T m = 58ºC, DG =22.5 kcal/mol) than the unmodified mrna6b. As a control, we synthesized another mrna that lacked a SD sequence but formed a secondary structure of similar stability (T m = 51ºC, DG =21.7 kcal/mol) (Figure 3B). Steady-state fluorescence of mrna6b was monitored by exciting cy3 at 550 nm and scanning for fluorescence emission at wavelengths nm. Fluorescence emission at wavelengths below 560 nm were not scanned in order to avoid the large excitation peak of cy3 at 550 nm and to minimize background due to light scattering. mrna6b showed very low cy3 emission (570 nm) but significant cy5 emission (660 nm) due to FRET from cy3 to cy5 (Figure 3A, blue trace). This indicated that the 5 0 and 3 0 ends of mrna6b were close to each other in solution. Addition of the 30S subunit to mrna6b resulted in a further 1.5-fold increase in cy5 emission, presumably due to the mrna binding to the 30S subunit. However, the cy3 emission remained low, indicating that the 5 0 and 3 0 ends of mrna6b remained close to each other. Remarkably, when fmet-trna fmet and IFs were added to the reaction, the fluorescence emission of cy3 increased significantly (4- to 5-fold), and the emission of cy5 consistently showed a slight decrease (Figure 3A, red trace). This indicated that the fluorescence emission of cy3 dye is no longer quenched by the cy5 dye, most likely because mrna6b has unfolded, thereby separating the two dyes. Consistent with this result, a similar decrease in FRET efficiency was observed when mrna6b was unfolded by the hybridization of a complimentary DNA oligonucleotide (Figure S2A). Thus, the 30S subunit with all three IFs and fmettrna fmet disrupts the secondary structure of mrna6b. The decrease in cy5 emission was not as significant, due to the increased quantum efficiency of the probe that occurs when the mrna unfolds. Interestingly, experiments done in parallel with the control mrna, which lacked the SD sequence, showed no significant increase in cy3 or decrease in cy5 emission intensity in the presence of the 30S subunit, fmet-trna fmet, and IFs (Figure 3B). This showed that the SD sequence was essential for disrupting the secondary structure of mrna6b. The interaction of cy3/cy5-labeled mrna6b with the translation initiation complex was next analyzed using a rapid kinetic technique. The initial binding of mrna6b with the 30S subunit was monitored by exciting cy3 and examining cy5 emission due to FRET or by exciting cy5 directly and examining cy5 emission. In both cases, a rapid increase in cy5 fluorescence emission was observed in the presence of the 30S subunit, and the change was completed in about 15 s (Figure 4A). This rapid increase in cy5 fluorescence was possibly due to the interaction of the 30S subunit with the loop region of mrna6b, as was observed with the pyrene-labeled

6 Molecular Cell 110 Figure 3. Unfolding of mrna6b (A) Secondary structure of mrna6b with cyanin 3 (cy3, blue star) and cyanin 5 (cy5, orange star) probes at the 5 0 and 3 0 ends, respectively (left panel). In the folded state, the cy3 and cy5 probes are close, resulting in high FRET efficiency. mrna6b in buffer (blue trace), mrna6b bound to the 30S subunit (green trace), or mrna6b bound to 30S subunit complexed with fmet-trna fmet and the three IFs (red trace). (B) Secondary structure of control mrna (no SD) that lacks the SD sequence (left panel). Control mrna (no SD) in buffer (blue trace), bound to the 30S subunit (green trace), and bound to the 30S subunit complexed with fmet-trna fmet and the three IFs (red trace). mrna6b. Excitation of cy3 caused cy5 emission to increase due to FRET, and the kinetic profile was similar when cy5 was directly excited. This showed that the 30S subunit interacted with mrna6b without separating the 5 0 and 3 0 ends. Remarkably, the association of mrna6b with the 30S subunit in the presence of fmet-trna fmet and all three IFs showed a different profile. When cy3 is excited and the emission of cy5 is monitored, the initial rapid increase in cy5 emission is followed by a slow decrease in cy5 emission (Figure 4B, magenta trace). Similarly, when cy3 was excited and emission of cy3 was monitored or when cy5 was excited and cy5 emission was monitored, two phases were observed (Figure 4B). The two phases were an initial rapid increase followed by a slower increase in fluorescence intensity (second phase, k 2 = s -1 ). The rapid first phase corresponded to the initial binding of mrna6b to the 30S complex. The increase in cy3 and cy5 emissions were likely due to the microenvironment of the 30S subunit. Even though the fluorescence emission of cy3 and cy5 increased when mrna6b associated with the 30S subunit, the FRET efficiency between the fluorophores decreased in the slow second phase. The decrease in FRET between cy3 and cy5 during the second phase indicated that the distance between the 5 0 and 3 0 ends of mrna6b had increased. The simplest interpretation of these results is that the 30S subunit complexed with fmet-trna fmet, and IFs disrupted the secondary structure of mrna6b, thereby separating the 5 0 and 3 0 ends of the mrna. In order to verify that the change in FRET was not due to the microenvironment of cy3, we synthesized mrna6b with only a cy3 label at the 5 0 end and monitored its emission when complexed with the 30S subunit, fmet-trna fmet, and IFs. During the time period when the second phase was normally observed, no change in cy3 emission intensity was detected (data not shown). This showed that the change in FRET efficiency between cy3 and cy5 was not due to changes in the photophysical properties of cy3 in the 30S complex but due to an increase in the distance between the donor and the acceptor dyes caused by unfolding of the mrna. IFs Important for mrna Unfolding and the Role of GTP We next investigated which IFs are essential for unfolding mrna6b and whether GTP is hydrolyzed in this process. Factor omission experiments showed that the minimal requirement for unfolding mrna6b by the 30S subunit was the presence of fmet-trna fmet and IF2 (Figures S2C S2F). Interestingly, IF3 slightly inhibited mrna unfolding, while IF1 promoted unfolding of the mrna. It is worth noting that these results agree with the results from the mrna dissociation experiments. The requirement for GTP hydrolysis was determined using GTP analogs. IF2 was complexed with GTP, GDPNP, GTP-gS, or GDP. A control without any nucleotide was also included (no NTP). The different 30S initiation complexes were excited at 550 nm (which excited cy3), and the fluorescence emission intensity of cy3 and cy5 was measured (Figure 4C). The fluorescence emission intensity of cy3 increased in complexes containing GTP, GTP-gS, and GDPNP. In contrast, complexes containing GDP or no NTP did not show the same increase in cy3 fluorescence as the GTP bound form of IF2. This suggested that the GTP bound form of IF2 was important but that GTP hydrolysis was not required for unfolding

7 Unfolding of Structured mrnas during Translation 111 Figure 4. Time Course of mrna6b Unfolding and the Role of GTP in Unfolding (A) Transient-state association kinetics of mrna6b with the 30S subunit. Cy3 was excited, and the fluorescence emission of cy5 due to FRET was measured (magenta trace), or cy5 was directly excited and the fluorescence emission of cy5 was measured (green trace) for a short time period (40 s). (B) Similar to (A), but the 30S subunit was complexed with fmet-trna fmet and IFs. The change in fluoresecence intensity was measured for a longer time period (200 s) to examine the slow second phase. In addition, cy3 was directly excited, and the fluorescence emission of cy3 was measured (blue trace). (C) Steady-state fluorescence measurement of mrna6b bound to the 30S subunit complexed with fmet-trna fmet and the three IFs in the presence of GTP (red trace), GTP-gS (blue trace), GDPNP (green trace), GDP (purple trace), or no GTP (cyan trace). (D) Hydrolysis of [a- 32 P]GTP by the 50S subunit (lane 1); 70S ribosome, IFs and fmet-trna fmet (lane 2); 30S subunit, IFs and fmet-trna fmet (lane 3); 30S subunit, mrna6b, IFs and fmet-trna fmet (lane 4); 30S subunit, mrna0 (polyu), IFs and fmet-trna fmet (lane 5); 30S subunit (lane 6); and IFs (lane 7). GTP was spotted as a marker (lane 8). The positions of GTP and GDP on the TLC are indicated. (E) Cartoon showing binding of a structured mrna by the bacterial initiation complex. In the first step, the folded mrna binds to the 30S subunit (blue) complexed with fmet-trna fmet, IF1, IF2, and IF3. In a subsequent step, the 30S complex unfolds the mrna and forms base pairs with the ASD sequence resulting in a stable 30S initiation complex. Shown in red is the ASD of 16S rrna. the mrna. It is known that the GTP bound form of IF2 has a higher affinity for the 30S subunit in the presence of fmet-trna fmet. In addition, IF2GTP is known to stabilize the binding of fmet-trna fmet to the 30S subunit (Antoun et al., 2003). These results were further verified by monitoring GTP hydrolysis directly using radiolabeled GTP (Figure 4D). No difference was observed in the amount of GTP hydrolyzed by the 30S initiation complex in the absence of mrna or in the presence of structured mrna6b or unstructured mrna0b (polyu) (w20%) (Figure 4D, compare lanes 3 5). These results are in agreement with the FRET-based unfolding assay, which showed that GTP hydrolysis was not essential for unfolding mrna. Indeed, studies have shown that GTP hydrolysis by IF2 occurs after 50S subunit association and is important for its dissociation from the ribosome (Antoun et al., 2003). In order to further establish that unfolding of the mrna by the 30S initiation complex was a passive process, we made use of the DNA oligonucleotide that is complimentary to mrna6b. Binding of the DNA

8 Molecular Cell 112 oligonucleotide to the mrna6b results in the unfolding of the mrna, thereby decreasing the FRET efficiency between the cy3 and cy5 probes. The rate of unfolding of mrna6b by the DNA oligonucleotide was only z6- fold slower (k on = mm 21 s 21 ) than the rate at which the 30S initiation complex disrupted the secondary structure of mrna6b. Since both the DNA oligonucleotide and the 30S initiation complex disrupt the secondary structure at approximately the same rate, it is consistent with the fact that unfolding of mrna by the initiation complex is a passive process (see Discussion). Codon-Anticodon Interaction Is Necessary for mrna Stabilization Since fmet-trna fmet was required for stabilization of the mrna on the 30S initiation complex, we wanted to investigate whether or not codon-anticodon interaction was also essential for this process. Therefore, we constructed an mrna identical to mrna6b but had the canonical AUG start codon replaced with CUG (mrna6b CUG). Association experiments with mrna6b (CUG) did not show a distinct second phase when fmettrna fmet was added to the 30SmRNAIFs complex (data not shown). In addition, no difference was observed in the dissociation rate constants for mrna6b (CUG) from the 30S subunit in the absence (k off = s 21 ) or presence of IFs and fmet-trna fmet (k off = s 21 )(Figure S1F). IF3 is known to promote the dissociation of fmettrna fmet from 30S initiation complexes containing codons other than the start codon in the P site (Gualerzi et al., 1971; Hartz et al., 1989, 1990; Sussman et al., 1996). Therefore, IF3 may inhibit the interaction between fmet-trna fmet and mrna6b (CUG) thereby preventing the unfolding of this mrna in the 30S pre-initiation complex. In order to test this idea, we examined mrna 6b (CUG) s stabilization in the 30SIF1IF2fMet-tRNA fmet complex. We did not observe any additional stabilization in the absence of IF3 (data not shown). These results show that the interaction between the anticodon of fmet-trna fmet and the start codon is important for stabilizing structured mrnas in the initiation complex. Discussion In cells, binding of mrna to the ribosome is kinetically driven because mrnas compete to form the translation initiation complex. mrnas that have fast association and slow dissociation kinetics form a stable initiation complex and are likely to be translated efficiently. Previous studies have shown that the 30S subunit can accommodate structured mrnas (Nivinskas et al., 1999; Ringquist et al., 1993; Sacerdot et al., 1998; Yusupova et al., 2001). In fact, the TIR of the mrna encoding threonyl synthetase, which forms a structured domain that is important for translational control, bound to the ribosome was recently solved by X-ray crystallography (Jenner et al., 2005). However, mrna6a, which is fully double-stranded, does not bind to the 30S subunit even in the presence of IFs and initiator trna. The inability of the 30S subunit to bind mrna6a may be due to the absence of single-stranded regions that act as standby sites for the 30S subunit to bind. In contrast, mrna6a (Ext) binds to the 30S subunit and unfolds in the presence of IFs and fmet-trna fmet to form a stable complex. Similarly, mrna6b, which also forms a stable hairpin, binds to the 30S subunit. It is possible that the 8 nt single-stranded loop present in mrna6b may facilitate interaction with the 30S subunit. Thus, the presence of single-stranded regions in mrna that act as standby sites are important for the 30S subunit to initially bind structured mrnas. Rapid interaction of the 30S subunits with singlestranded regions in mrna is consistent with the standby model (de Smit and van Duin, 2003). mrnas may initially bind to the small subunit protein S1, which has been proposed to play an important role during translation initiation by interacting with mrna (Sorensen et al., 1998; Subramanian, 1983). S1 is an essential protein of the OB fold family that has high affinity for single-stranded RNA (Draper and Reynaldo, 1999; Draper and von Hippel, 1979). S1 is located at the junction of the head and the platform domain on the solvent side of the 30S subunit (Sengupta et al., 2001). This location allows interaction with mrna sequences upstream of the SD region. Interestingly, S1 binds with high affinity to pseudoknot structures that have a purine-rich 7 nt loop (Ringquist et al., 1995). mrna6b and mrna6c have 8 nt purine rich loops. Therefore, it is possible that, in the first step, S1 binds the purine-rich loop within these structured mrnas and tethers the mrnas on the 30S subunit. In a second step, our data show that further stabilization of the structured mrna on the 30S subunit requires the participation of IFs and fmet-trna fmet. A possible insight into the stabilization of structured mrna on the 30S subunit can be found by analyzing the mrna dissociation rate constants. Comparing the dissociation rate constants of mrna6b and mrna6c (mrnas with identical SD sequence) shows that mrna6b dissociates 10-fold faster than mrna6c (Table 2). The secondary structure of mrna6b is more stable than mrna6c, suggesting that a competing secondary structure in the mrna may inhibit the SD sequence from interacting with the ASD sequence, thereby accelerating the dissociation of the mrna from the 30S subunit. Surprisingly, in the presence of IFs and fmet-trna fmet, the dissociation rates of mrna6b and mrna6c are similar, suggesting that IFs and fmet-trna fmet may promote the interaction between the SD-ASD sequences to form a stable 30S initiation complex. Once a stable SD-ASD interaction is established, then disruption of the 6 bp minihelix becomes rate limiting for the dissociation of mrna6b and mrna6c, resulting in similar dissociation rate constants. The molecular basis for the switch in the activity of the 30S subunit is not known. One possibility is that the IFs promote the interaction of fmet-trna fmet with the start codon of the mrna causing the mrna to unfold on the 30S subunit. Since the kinetics of mrna folding are rapid, it is important for the SD sequence to be in close proximity to the ASD sequence. Proper positioning of the SD sequence may be accomplished by the interaction of fmet-trna fmet with the authentic start codon in the mrna. As the mrna unfolds spontaneously, the SD sequence base pairs with 16S rrna, thereby driving the equilibrium toward the unfolded state and trapping the mrna on the 30S initiation complex. In agreement

9 Unfolding of Structured mrnas during Translation 113 with this model, we are unable to observe stabilization of mrna6b (CUG), suggesting that interaction with fmettrna fmet is critical for stably binding the structured mrna. Furthermore, we show that the presence of an SD sequence in the mrna is also important for unfolding the mrna. Thus, the interaction of the 30S initiation complex with structured mrnas occurs in at least two steps: a rapid initial binding step followed by an unfolding step induced by IFs and fmet-trna fmet that facilitates SD-ASD interaction and stabilizes the mrna on the 30S initiation complex (Figure 4E). Structural studies showed that binding of IF1 (Carter et al., 2001), IF2 (Allen et al., 2005; Wakao et al., 1991), and IF3 (McCutcheon et al., 1999; Pioletti et al., 2001) triggered conformational changes on the 30S subunit. These conformational changes induced in the 30S subunit by IFs and fmet-trna fmet may promote codon-anticodon interaction and SD-ASD interaction (Ehresmann et al., 1986; Gualerzi and Pon, 1990; La Teana et al., 1996; van Dieijen et al., 1976; Van Duin et al., 1976), thereby helping the structured mrna to unfold on the 30S subunit. Our results show that unfolding of structured mrnas does not require GTP hydrolysis. Therefore, the thermodynamic driving force for unfolding the mrna probably comes from the codon-anticodon interaction and the base pairs that form between the SD and ASD sequences. A DNA oligonucleotide that is fully complimentary to mrna6b is able to efficiently unfold the mrna to form a duplex. However, a short DNA oligonucleotide that corresponds to the ASD of 16S rrna is unable to melt the secondary structure of mrna6b (Figure S2A). This short DNA oligonucleotide cannot interact with the single-stranded loop of mrna6b; therefore, it is unable to initiate unfolding of mrna6b. Presuambly, the single-stranded loop of mrna6b allows the fully complimentary DNA oligonucleotide to initiate base pair formation and remain associated with the mrna resulting in helix formation. Although parallels can be drawn between the bacterial and eukaryotic initiation mechanisms, the requirements for unfolding mrna by the bacterial initiation complex are distinct from the eukaryotic process. In eukaryotes, specific IFs unwind mrna secondary structure in an ATP-dependent process to create an unstructured region that allows the 43S initiation complex to bind an mrna (Kapp and Lorsch, 2004). In contrast, we show that bacterial initiation requires the participation of the 30S subunit complexed with fmet-trna fmet and IF2GTP to stably bind structured mrnas that possess a SD sequence and an authentic start codon. Moreover, energy from NTP hydrolysis is not required for binding structured mrnas. The ability of bacterial initiation complexes to use such a simple mechanism to unfold mrna structures may have been the evolutionary forerunner to the more specialized unwinding mechanism found in eukaryotic translation. Experimental Procedures Preparation of 30S, trna, mrna, and IFs Ribosomal 30S subunits were isolated from E. coli MRE 600 cells (Powers and Noller, 1991). Aminoacylation of E. coli trna fmet was performed using purified E. coli methionyl synthetase (Studer et al., 2003), formylated using methionyl transformylase, and purified by HPLC (Odom et al., 1988). mrnas containing the cy3/cy5 dyes or a3 0 amino linker were purchased from Dharmacon and derivatized with pyrene (Studer et al., 2003). IFs were purified as described (Shimizu et al., 2001) and their activity determined as described (Beaudry et al., 1979; Petrelli et al., 2001). IF2-dependent GTP hydrolysis assays were performed using [a- 32 P]GTP, analyzed on PEI cellulose TLC (Schwemmle and Staeheli, 1994), and quantitated using a phosphorimager (Molecular Dynamics). Native Gel and Thermal Melting Experiments [ 32 P]-labeled mrnas ( cpm) were analyzed on 15% native polyacrylamide gel containing 40 mm Tris acetate and 12 mm MgCl 2 (Feinberg and Joseph, 2001). Thermal melting experiments (Cole et al., 1972) were performed on a Beckman Coulter DU-640 spectrophotometer and analyzed using Meltwin Software. mrna Association The 30S ribosomal subunits (0.25 mm) were heat activated at 42ºC for 10 min in polyamine buffer (Bartetzko and Nierhaus, 1988) (20 mm HEPES-KOH [ph 7.6], 6 mm magnesium chloride, 150 mm ammonium chloride, 4 mm 2-mercaptoethanol, 0.05 mm spermine, and 2 mm spermidine) containing 1mM GTP (or GTP analog), slow cooled to 37ºC, and incubated for 10 min at 37ºC. Depending on the experiment, IFs (0.5 mm) and fmet-trna fmet (0.75 mm) were incubated together for 10 min at 37ºC. IFs in the presence or absence of fmet-trna fmet were then added to the 30S subunits and allowed to incubate at 37ºC for 20 min. The appropriate dye-labeled mrna (0.2 mm) was incubated at 37ºC for 30 min in polyamine buffer containing 1 mm GTP (or GTP analog). The complexes were then placed at 25ºC for at least 5 min prior to performing mrna association experiments. All concentrations indicated are final concentrations before mixing. mrna Dissociation The 30S complexes were formed as described above for the mrna association experiments, except that heat activated 30S ribosomal subunits (0.25 mm) were mixed with the appropriate dye-labeled mrna (0.2 mm) and incubated at 37ºC for 10 min to preform the 30SmRNA complex. IFs (0.5 mm) and fmet-trna fmet (0.75 mm) were incubated together for 10 min at 37ºC in polyamine buffer with 1 mm GTP. Experiments with individual factors were performed at both 0.5 mm and 5 mm final concentration of IF1 and IF3. IFs, with or without fmet-trna fmet, were then added to the 30S subunits and allowed to incubate at 37ºC for 20 min. Longer incubation times did not change the dissociation rate constants, indicating that equilibrium has been achieved. The 30S initiation complexes were then placed at 25ºC for at least 5 min prior to measuring the dissociation kinetics. Excess unlabeled mrna (2.5 mm) was used as a trap. The unlabeled mrna in polyamine buffer containing 1 mm GTP was incubated at 37ºC for 30 min, then placed at 25ºC for at least 5 min prior to mixing with the 30S complex containing the dye-labeled mrna. Steady-State Fluorescence Measurements The fluorescence intensity of the 30SmRNA complex (0.25 mm in 200 ml final volume) prepared as described above was measured at 25ºC with a photon-counting fluorometer (Fluoromax-P, JY Horiba). Complexes containing pyrene-labeled mrnas were excited at 343 nm, and the emission spectrum from nm wavelength was recorded. Complexes containing cy3/cy5-labeled mrnas were excited at 550 nm (cy3) or 650 nm (cy5), and the emission spectrum from nm wavelength were recorded. The emission maxima for cy3 and cy5 dyes are 570 nm and 670 nm, respectively. To determine which factor caused the unwinding of the mrna, 30S subunit and mrna were incubated at 25ºC for 1 min, and then each factor was individually added and allowed to incubate with the 30SmRNA complex for approximately 45 min prior to an emission scan. Kinetic Association and Dissociation Experiments Rapid kinetic experiments were performed at 25ºC on a stoppedflow instrument (msfm-20, BioLogic, France). The complexes were prepared as described above and mixed at 1:1 ratio (mixing volume was 107 ml). The excitation wavelength was 343 nm (band pass

10 Molecular Cell nm) and the fluorescence emission was measured after passing a long-pass filter 361 AELP (Omega Optical) installed in front of the detector. For complexes containing cy3/cy5-labeled mrnas, the excitation wavelength was 550 nm (cy3) or 650 nm (cy5) (band pass 10 nm), and the fluorescence emission was measured after passing a long-pass filter 3RD/ (cy3 emission) or a 3RD/ 670LP (cy5 emission) with light blocker 3rdM/B15 (Omega Optical). The association experiment for each mrna was performed at five different mrna concentrations (0.18, 0.36, 0.56, 0.75, and 1.2 mm before mixing) with fixed concentration of the 30S complex (0.25 mm before mixing), and each experiment was repeated at least twice. In the case of mrna6a (Ext), the concentration of the mrna was 0.36 mm before mixing. The association data measured under second-order conditions were analyzed by nonlinear regression using the DynaFit program (Kuzmic, 1996). The progress curves were fitted to a two-step reaction mechanism represented by a set of chemical equations written symbolically by the scheme: 30S + mrna <===> 30SmRNA <===> 30SmRNA* in DynaFit. Step 1 is the bimolecular association of mrna with the 30S subunit with rate constants k 1 and k 21. In step 2, the mrna unfolds with rate constants k 2 and k 22. The program determines the approporiate differential equations for the reaction mechanism and then uses iterative nonlinear regression analysis to determine the best-fit parameters for the experimental data. Several alternate reaction mechanisms were analyzed using DynaFit. Statistical criteria and visual inspection of the residual distribution plots were used to obtain the best fits. The association experiment for each mrna was performed at five different mrna concentrations (0.18, 0.36, 0.56, 0.75, and 1.2 mm before mixing), and each experiment was repeated at least twice. The kinetic traces were independently fitted. All dissociation curves were analyzed by least squares fitting to a one-phase exponential decay equation using GraphPad Prism software (San Diego, California). Supplemental Data Supplemental Data include two figures and one table and can be found with this article online at content/full/22/1/105/dc1/. Acknowledgments We thank Yitzhak Tor for the use of his spectrophotometer to perform RNA melting studies; Uttam RajBhandary for the MTF clone; Takuya Ueda for the vectors containing IF1, 2, and 3 genes; Jack Kyte, John Wheeler, Joseph Adams, and Mikhail Levin for advice on curve fitting; and David Draper, Gourishankar Ghosh, Partho Ghosh, Joseph Adams, and Steven Phelps for comments on the manuscript. This work was supported by grants from the NIH (R01 GM to S.J), NSF (MCB to S.J.), and HFSP (RGY16 to S.J.) and a predoctoral training grant (to S.M.S). Received: November 11, 2005 Revised: January 25, 2006 Accepted: February 7, 2006 Published: April 6, 2006 References Allen, G.S., Zavialov, A., Gursky, R., Ehrenberg, M., and Frank, J. (2005). The Cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121, Antoun, A., Pavlov, M.Y., Andersson, K., Tenson, T., and Ehrenberg, M. (2003). The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis. EMBO J. 22, Bartetzko, A., and Nierhaus, K.H. (1988). Mg2+/NH4+/polyamine system for polyuridine-dependent polyphenylalanine synthesis with near in vivo characteristics. Methods Enzymol. 164, Beaudry, P., Sander, G., Grunberg-Manago, M., and Douzou, P. (1979). Cation-induced regulatory mechanism of GTPase activity dependent on polypeptide initiation factor 2. Biochemistry 18, Boelens, R., and Gualerzi, C.O. (2002). Structure and function of bacterial initiation factors. Curr. Protein Pept. Sci. 3, Canonaco, M.A., Gualerzi, C.O., and Pon, C.L. (1989). Alternative occupancy of a dual ribosomal binding site by mrna affected by translation initiation factors. Eur. J. Biochem. 182, Carter, A.P., Clemons, W.M., Jr., Brodersen, D.E., Morgan-Warren, R.J., Hartsch, T., Wimberly, B.T., and Ramakrishnan, V. (2001). Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Science 291, Cole, P.E., Yang, S.K., and Crothers, D.M. (1972). Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. Biochemistry 11, de Smit, M.H. (1998). Translational Control by mrna Structure in Eubacteria: Molecular Biology and Physical Chemistry (New York, New York: CSHL Press). de Smit, M.H., and van Duin, J. (2003). Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mrna. J. Mol. Biol. 331, Draper, D. (1993). Mechanism of Translational Initiation and Repression in Prokaryotes (New York, New York: Plenum Press). Draper, D.E., and Reynaldo, L.P. (1999). RNA binding strategies of ribosomal proteins. Nucleic Acids Res. 27, Draper, D.E., and von Hippel, P.H. (1979). Interaction of Escherichia coli ribosomal protein S1 with ribosomes. Proc. Natl. Acad. Sci. USA 76, Ehresmann, C., Moine, H., Mougel, M., Dondon, J., Grunberg-Manago, M., Ebel, J.P., and Ehresmann, B. (1986). Cross-linking of initiation factor IF3 to Escherichia coli 30S ribosomal subunit by transdiamminedichloroplatinum(ii): characterization of two cross-linking sites in 16S rrna; a possible way of functioning for IF3. Nucleic Acids Res. 14, Feinberg, J.S., and Joseph, S. (2001). Identification of molecular interactions between P site trna and the ribosome essential for translocation. Proc. Natl. Acad. Sci. USA 98, Gualerzi, C.O., and Pon, C.L. (1990). Initiation of mrna translation in prokaryotes. Biochemistry 29, Gualerzi, C., Pon, C.L., and Kaji, A. (1971). Initiation factor dependent release of aminoacyl-trnas from complexes of 30S ribosomal subunits, synthetic polynucleotide and aminoacyl trna. Biochem. Biophys. Res. Commun. 45, Hartz, D., McPheeters, D.S., and Gold, L. (1989). Selection of the initiator trna by Escherichia coli initiation factors. Genes Dev. 3, Hartz, D., Binkley, J., Hollingsworth, T., and Gold, L. (1990). Domains of initiator trna and initiation codon crucial for initiator trna selection by Escherichia coli IF3. Genes Dev. 4, Hoe, N.P., and Goguen, J.D. (1993). Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated. J. Bacteriol. 175, Holder, J.W., and Lingrel, J.B. (1975). Determination of secondary structure in rabbit globin messenger RNA by thermal denaturation. Biochemistry 14, Jenner, L., Romby, P., Rees, B., Schulze-Briese, C., Springer, M., Ehresmann, C., Ehresmann, B., Moras, D., Yusupova, G., and Yusupov, M. (2005). Translational operator of mrna on the ribosome: how repressor proteins exclude ribosome binding. Science 308, Johansson, J., Mandin, P., Renzoni, A., Chiaruttini, C., Springer, M., and Cossart, P. (2002). An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, Kapp, L.D., and Lorsch, J.R. (2004). The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, Kuzmic, P. (1996). Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal. Biochem. 237, La Teana, A., Gualerzi, C.O., and Brimacombe, R. (1995). From stand-by to decoding site. Adjustment of the mrna on the 30S ribosomal subunit under the influence of the initiation factors. RNA 1,