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The Cambridge handbook of forensic psychology
\"Forensic psychology has developed and extended from an original, narrow focus on presenting evidence to the courts to a wider application across the whole span of civil and criminal justice, which includes dealing with suspects, offenders, victims, witnesses, defendants, litigants and justice professionals. This handbook provides an encyclopedic-style source regarding the major concerns in forensic psychology. It is an invaluable reference text for practitioners within community, special hospital, secure unit, prison, probation and law enforcement forensic settings, as well as being appropriate for trainees and students in these areas. It will also serve as a companion text for lawyers and psychiatric and law enforcement professionals who wish to be apprised of forensic psychology coverage. Each entry provides a succinct outline of the topic, describes current thinking, identifies relevant consensual or contested aspects and alternative positions. Readers are presented with key issues and directed towards specialized sources for further reference\"-- Provided by publisher.
Book
Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding
A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex
1
–
3
. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12–14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed
4
–
6
. Here we present cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2—universal structural features of RNAP—in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life.
Cryo-electron microscopy structures of bacterial RNAP–promoter DNA complexes, including structures of partially melted intermediates, suggest a universally conserved common mechanism for promoter DNA opening prior to gene expression.
Journal Article
Basis of narrow-spectrum activity of fidaxomicin on Clostridioides difficile
Fidaxomicin (Fdx) is widely used to treat
Clostridioides difficile
(
Cdiff
) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown.
Cdiff
infections are a leading cause of nosocomial deaths
1
. Fidaxomicin, which inhibits RNA polymerase, targets
Cdiff
with minimal effects on gut commensals, reducing recurrence of
Cdiff
infection
2
,
3
. Here we present the cryo-electron microscopy structure of
Cdiff
RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of
Cdiff
RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in
Cdiff
RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.
Structural analysis of
Clostridioides difficile
RNA polymerase in complex with fidaxomicin combined with biochemical, genetic and bioinformatic analyses identifies a key residue that determines fidaxomicin sensitivity.
Journal Article
Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin
We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739–751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.
Journal Article
Structural basis for backtracking by the SARS-CoV-2 replication–transcription complex
Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA–protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3′ segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3′ end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.
Journal Article
Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication–transcription complex
The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication–transcription complex or RTC) associated with two copies of nsp13 (nsp13
2
–RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp13
2
–RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp13
2
–RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.
In their complex, the SARS-CoV-2 nsp13 helicase and RNA polymerase would translocate on RNA in opposite directions. Cryo-EM and MD simulations resolve this conundrum, suggesting an allosteric mechanism to turn the helicase on and off.
Journal Article
Phage T7 Gp2 inhibition of Escherichia coli RNA polymerase involves misappropriation of σ⁷⁰ domain 1.1
Bacteriophage T7 encodes an essential inhibitor of the Escherichia coli host RNA polymerase (RNAP), the product of gene 2 (Gp2). We determined a series of X-ray crystal structures of E. coli RNAP holoenzyme with or without Gp2. The results define the structure and location of the RNAP σ ⁷⁰ subunit domain 1.1 [Formula] inside the RNAP active site channel, where it must be displaced by the DNA upon formation of the open promoter complex. The structures and associated data, combined with previous results, allow for a complete delineation of the mechanism for Gp2 inhibition of E. coli RNAP. In the primary inhibition mechanism, Gp2 forms a protein–protein interaction with [Formula], preventing the normal egress of [Formula] from the RNAP active site channel. Gp2 thus misappropriates a domain of the RNAP holoenzyme, [Formula], to inhibit the function of the enzyme.
Journal Article
Structural Insights into De Novo Promoter Escape by Mycobacterium tuberculosis RNA Polymerase
Transcription in bacteria is a multi-step process. In the first step, contacts between RNA polymerase and the promoter DNA must be established for transcription initiation to begin, but then these contacts must be broken for the enzyme to transition into the elongation phase. Single-molecule and biochemical observations report that promoter escape is a highly regulated and sometimes rate-limiting step in the transcription cycle; however, the structural mechanisms of promoter escape remain obscure. Promoter escape also serves as the target for the clinically important antibiotic rifampicin, used to treat tuberculosis. Here, we present seven distinct intermediates showing the structural details of
M. tuberculosis
RNA polymerase initial transcribing complexes and promoter escape, using a de novo cryo-electron microscopy approach. We describe the structural rearrangements that RNA polymerase undergoes to clear the promoter, including those required to release the initiation factor, σ, providing a structural account for decades of biochemical observations. These structures and supporting biochemistry provide a model of promoter escape, a universal step in the transcription cycle, with conformations that may be used to develop Rifampicin alternatives.
Promoter escape is a key step in bacterial transcription and a target of the antibiotic rifampicin. Here, the authors use cryo-EM to explore this step, finding seven structural intermediates of
M. tuberculosis
RNA polymerase during promoter escape.
Journal Article
Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures
The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a
Mycobacterium smegmatis
(
Msm
) open promoter complex (RPo), along with structural analysis of the
Msm
RPo and a previously reported 2.76 Å-resolution crystal structure of an
Msm
transcription initiation complex with a promoter DNA fragment. We observe the interaction of the
Msm
RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the αCTD may play a role in
Mtb
transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP β′ subunit. Finally, our analysis reveals the disposition of the N-terminal segment of
Msm
σ
A
, which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with
E. coli
.
Understanding of the mycobacterial transcription system is useful to the development of therapeutics against tuberculosis infection. Here the authors present the crystal structure of a complete
M. smegmatis
RNA polymerase open promoter complex that reveals unique features of the mycobacterial polymerase.
Journal Article