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Rui Sousa

Publications and source records attributed to Rui Sousa.

17 recordsLinked to original sources
PubMed2006

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1.

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.

Adenosine Triphosphate↗
PubMed2006

Keep the traffic moving: mechanism of the Hsp70 motor.

Hsp70s are ubiquitous chaperones that use ATP hydrolysis to drive a variety of protein processing reactions, including a number of steps in protein trafficking. Recent studies have shed light on how ATP might generate conformational changes in an Hsp70 molecule and how such changes might be harnessed to drive processes as diverse as protein import into subcellular organelles and uncoating of clathrin-coated vesicles.

Animals↗
PubMed2006

Translocation by T7 RNA polymerase: a sensitively poised Brownian ratchet.

Studies of halted T7 RNA polymerase (T7RNAP) elongation complexes (ECs) or of T7RNAP transcription against roadblocks due to DNA-bound proteins indicate that T7RNAP translocates via a passive Brownian ratchet mechanism. Crystal structures of T7RNAP ECs suggest that translocation involves an active power-stroke. However, neither solution studies of halted or slowed T7RNAP ECs, nor crystal structures of static complexes, are necessarily relevant to how T7RNAP translocates during rapid elongation. A recent single molecule study of actively elongating T7RNAPs provides support for the Brownian ratchet mechanism. Here, we obtain additional evidence for the existence of a Brownian ratchet during active T7RNAP elongation by showing that both rapidly elongating and halted complexes are equally sensitive to pyrophosphate. Using chemical nucleases tethered to the polymerase we achieve sub-ångström resolution in measuring the average position of halted T7RNAP ECs and find that the positional equilibrium of the EC is sensitively poised between pre-translocated and post-translocated states. This may be important in maximizing the sensitivity of the polymerase to sequences that cause pausing or termination. We also confirm that a crystallographically observed disorder to order transition in a loop formed by residues 589-612 also occurs in solution and is coupled to pyrophosphate or NTP release. This transition allows the loop to make interactions with the DNA that help stabilize the laterally mobile, ligand-free EC against dissociation.

Biological Transport↗
PubMed2005

Crystallization of a functionally intact Hsc70 chaperone.

Hsp70s are essential chaperones with roles in a variety of cellular processes and representatives in all kingdoms of life. They are comprised of a nucleotide-binding domain (NBD) and a protein substrate-binding domain (SBD). Structures of isolated NBDs and SBDs have been reported but, until recently, a functionally intact Hsp70 containing both the NBD and SBD has resisted structure determination. Here, it is reported that preparation of diffraction-quality crystals of functionally intact bovine Hsc70 required (i) deletion of part of the protein to reduce oligomerization, (ii) point mutations in the interface between the SBD and NBD and (iii) use of high concentrations of the structure-stabilizing agents glycerol and trimethylamine oxide (TMAO). The introduction of point mutations in interdomain interfaces and the use of the potent structure stabilizer TMAO may be generally useful in crystallization of multidomain proteins that exhibit interdomain motions.

Alanine↗
PubMed2005

Structural basis of interdomain communication in the Hsc70 chaperone.

Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. We report here the 2.6 angstroms structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds.

Adenosine Triphosphate↗
PubMed2005

Major conformational changes during T7RNAP transcription initiation coincide with, and are required for, promoter release.

During transcription initiation conformational changes in the transcriptional machinery are required to accommodate the growing RNA, to allow the polymerase to release the promoter, and to endow the elongation complex with high processivity. In T7 RNA polymerase these changes involve refolding and reorientation of elements of the N-terminal domain, as well as changes in how the DNA is bound within the complex. However, when and where these conformational changes occur is unknown, and the role of these changes in allowing the polymerase to disengage the promoter is poorly understood. To address this we have used chemical nucleases tethered to the polymerase to monitor conformational changes, and engineered disulfide bonds to block conformational changes at defined steps in transcription. We find that many of the major structural transitions occur cooperatively, at a point coincident with promoter release. Moreover, promoter release requires that two elements of the polymerase which form a continuous promoter recognition surface in the initial transcription complex move apart: if this movement is blocked the polymerase cannot disengage the promoter.

DNA-Directed RNA Polymerases↗
PubMed2005

Weakening of the T7 promoter-polymerase interaction facilitates promoter release.

During transcription initiation, RNA polymerases retain interactions with their promoters until the RNA is extended to 8-13 nucleotides, at which point the polymerase releases the promoter and moves downstream. It has been shown that release of the T7 promoter is inhibited when the T7 RNA polymerase-promoter interaction is strengthened. Here we asked whether release would be facilitated when the T7 promoter-polymerase interaction is weakened by the introduction of promoter mutations known to reduce promoter activity. Using chemical and enzymatic probes to monitor the position of the polymerase as a function of RNA length, we found that promoter mutations upstream of -4 facilitated release of the polymerase from the promoter, but more downstream mutations did not have such effects. We also found that released complexes turn over more slowly than promoter-bound complexes, indicating that retention of promoter interactions contributes to the dissociation of short RNAs during initial transcription.

Bacteriophage T7↗
PubMed2005

Machinations of a maxwellian demon.

The mechanisms by which RNA polymerase moves along DNA during elongation have been difficult to determine experimentally. In this issue of Cell, Bar-Nahum et al. (2005) show that back and forth sliding of RNA polymerase on DNA may be coupled to bending of an alpha helix, which can alternately occlude and expose the NTP binding site. Transcription factors can regulate elongation by modulating this bending motion.

DNA↗
PubMed2004

Multiple roles for the T7 promoter nontemplate strand during transcription initiation and polymerase release.

Transcription initiation begins with recruitment of an RNA polymerase to a promoter. Polymerase-promoter interactions are retained until the nascent RNA is extended to 8-12 nucleotides. It has been proposed that accumulation of "strain" in the transcription complex and RNA displacement of promoter-polymerase interactions contribute to releasing the polymerase from the promoter, and it has been further speculated that too strong a promoter interaction can inhibit the release step, whereas a weak interaction may facilitate release. We examined the effects of partial deletion of the nontemplate strand on release of T7 RNA polymerase from the T7 promoter. T7 polymerase will initiate from such partially single-stranded promoters but binds them with higher affinity than duplex promoters. We found that release on partially single-stranded promoters is strongly inhibited. The inhibition of release is not due to an indirect effect on transcription complex structure or loss of specific polymerase-nontemplate strand interactions, because release on partially single-stranded templates is recovered if the interaction with the promoter is weakened by a promoter base substitution. This same substitution also appears to allow the polymerase to escape more readily from a duplex promoter. Our results further suggest that template-nontemplate strand reannealing drives dissociation of abortive transcripts during initial transcription and that loss of interactions with either the nontemplate strand or duplex DNA downstream of the RNA lead to increased transcription complex slippage during initiation.

DNA-Directed RNA Polymerases↗
PubMed2003

Discontinuous movement and conformational change during pausing and termination by T7 RNA polymerase.

Time-resolved characterization of T7 RNA polymerase pausing and terminating at a class II termination site has been carried out using site-specifically tethered chemical nucleases. The data indicate that T7RNAP normally moves uniformly down the template as a rigid body. However, at the class II site this movement is interrupted, and the leading edge of the polymerase moves further along the DNA than the trailing edge. This discontinuous movement may persist until it can no longer be accommodated by conformational changes in the elongation complex, at which point the polymerase can either pause or terminate. Termination, but not pausing, is abrogated by introduction of a disulfide bond between the polymerase fingers and thumb subdomains. The introduced cysteines disrupt a thumb-fingers salt-bridge and, under reducing conditions, this mutant enzyme shows reduced processivity coincident with extension of the RNA to 5 nt. These observations suggest that termination requires that the thumb and fingers subdomains move apart, in a reversal of a conformational change important for initially forming a stable transcription complex.

Bacteriophage T7↗
PubMed2003

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

Amino Acid Sequence↗
PubMed2003

Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions.

During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP). Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches.

Journal Article↗
PubMed2003

T7 RNA polymerase.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed2002

A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs.

A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2'-deoxy, 2'-NH2 and 2'-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2'-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2'-substituents (2'-OMe and 2'-N3), and may also enhance use of NTPs with modifications at other than the 2'-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.

Amino Acid Substitution↗
PubMed2002

Structural transitions mediating transcription initiation by T7 RNA polymerase.

During transcription initiation, RNA polymerases appear to retain promoter interactions while transcribing short RNAs that are frequently released from the complex. Upon transition to elongation, the polymerase releases promoter and forms a stable elongation complex. Little is known about the changes in polymerase conformation or polymerase:DNA interactions that occur during this process. To characterize the transitions that occur in the T7 RNA polymerase transcription complex during initiation, we prepared enzymes with Fe-BABE conjugated at 11 different positions. Addition of H(2)O(2) to transcription complexes prepared with these enzymes led to nucleic acid strand scission near the conjugate. Changes in the cleavage sites revealed a series of conformational changes and rearrangements of protein:nucleic acid contacts that mediate progression through the initiation reaction.

Binding Sites↗
PubMed2002

Role of T7 RNA polymerase His784 in start site selection and initial transcription.

The role of steric constraints vs sequence preference in start site selection by T7 RNA polymerase was investigated by using a series of synthetic promoters in which the preferred template strand 'CC' initiation sequence was moved away from its normal position relative to the -17 to -6 element of the T7 promoter. It was found that the CC sequence directs efficient initiation if placed 1 or 2 nt downstream of its normal position, but not if placed upstream, or more than 2 nt downstream, of +1. Mutagenesis revealed that part of the bias to initiate with GTP is due to an interaction between histidine 784 and the 2-amino group of a guanosine bound in the initiating triphosphate position. This interaction is also important for holding short transcripts within the transcription complex during initial transcription.

Alanine↗