DNA surface exploration and operator bypassing during target search.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 µs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

Spatiotemporal kinetics of the SRP pathway in live E. coli cells.

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state.

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA's are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.

Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI.

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Substrate specificity of the pseudouridine synthase RluD in Escherichia coli.

Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. We have analyzed substrate specificity of RluD in vivo. Mutational analyses have revealed: (a) RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of one U by C has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other); (c) A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation. Pseudouridines were determined in the ribosomal particles from a ribosomal large subunit defective strain (RNA helicase DeaD(-)). An absence of pseudouridines in the assembly precursor particles suggests that RluD directed isomerization of uridines occurs as a late step during the assembly of the large ribosomal subunit.

Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli.

Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as 'burst' kinetics on fluorescent polymeric model substrates.

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88+/-10, 42+/-10 and 34+/-2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA.

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.

Pseudouridylation of 23S rRNA helix 69 promotes peptide release by release factor RF2 but not by release factor RF1.

Pseudouridine [Ψ] is a frequent base modification in the ribosomal RNA [rRNA] and may be involved in the modulation of the conformational flexibility of rRNA helix-loop structures during protein synthesis. Helix 69 of 23S rRNA contains pseudouridines at the positions 1911, 1915 and 1917 which are formed by the helix 69-specific synthase RluD. The growth defect caused by the lack of RluD can be rescued by mutations in class I release factor RF2, indicating a role for helix 69 pseudouridines in translation termination. We investigated the role of helix 69 pseudouridines in peptide release by release factors RF1 and RF2 in an in vitro system consisting of purified components of the Escherichia coli translation apparatus. Lack of all three pseudouridines in helix 69 compromised the activity of RF2 about 3-fold but did not significantly affect the activity of RF1. Reintroduction of pseudouridines into helix 69 by RluD-treatment restored the activity of RF2 in peptide release. A Ψ-to-C substitution at the 1917 position caused an increase in the dissociation rate of RF1 and RF2 from the postrelease ribosome. Our results indicate that the presence of all three pseudouridines in helix 69 stimulates peptide release by RF2 but has little effect on the activity of RF1. The interactions around the pseudouridine at the 1917 position appear to be most critical for a proper interaction of helix 69 with release factors.

Ribosomal intersubunit bridge B2a is involved in factor-dependent translation initiation and translational processivity.

Intersubunit bridges are important for holding together subunits in the 70S ribosome. Moreover, a number of intersubunit bridges have a role in modulating the activity of the ribosome during translation. Ribosomal intersubunit bridge B2a is formed by the interaction between the conserved 23S rRNA helix-loop 69 (H69) and the top of the 16S rRNA helix 44. Within the 70S ribosome, bridge B2a contacts translation factors and the A-site tRNA. In addition to bridging the subunits, bridge B2a has been invoked in a number of other ribosomal functions from initiation to termination. In the present work, single-nucleotide substitutions were inserted at positions 1912 and 1919 of Escherichia coli 23S rRNA (helix 69), which are involved in important intrahelical and intersubunit tertiary interactions in bridge B2a. The resulting ribosomes had a severely reduced activity in a cell-free translation elongation assay, but displayed a nearly wild-type-level peptidyl transferase activity. In vitro reassociation efficiency decreased with all of the H69 variant 50S subunits, but was severest with the A1919C and DeltaH69 variants. The mutations strongly affected initiation-factor-dependent 70S initiation complex formation, but exhibited a minor effect on the nonenzymatic initiation process. The mutations decreased ribosomal processivity in vitro and caused a progressive depletion of 50S subunits in polysomal fractions in vivo. Mutations at position 1919 decreased the stability of a dipeptidyl-tRNA in the A-site, whereas the binding of the dipeptidyl-tRNA was rendered more stable with 1912 and DeltaH69 mutations. Our results suggest that the H69 of 23S rRNA functions as a control element during enzymatic steps of translation.

Synergistic cellulose hydrolysis can be described in terms of fractal-like kinetics.

A fractal-like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme-to-substrate ratios showed an apparent substrate inhibition consistent with a case where two-dimensional (2-D) surface diffusion of the cellobiohydrolase is rate-limiting. The action of Cel7A core and Cel5A was instead consistent with a three-dimensional (3-D) diffusion-based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme-to-substrate ratio, but this effect was gradually reduced at lower enzyme-to-substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal-like kinetics analogue of a first-order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions.