Recombinant Pongo abelii Transcription elongation factor SPT5 (SUPT5H), partial

Basic Understanding and Fundamental Functions

• What is the primary function of SPT5/SUPT5H in transcription elongation?

  SPT5 functions as a key transcription elongation factor that both positively and negatively regulates RNA polymerase II (Pol II) activity. It forms a heterodimer with SPT4, known as the DSIF complex (DRB sensitivity-inducing factor in humans), which initially induces promoter-proximal pausing of Pol II and subsequently promotes productive elongation after phosphorylation .

  Mechanistically, SPT5 interacts extensively with the clamp, protrusion, and wall domains of RNA Pol II, effectively locking the clamp in a closed conformation that encases the DNA template in the central cleft . This conformation is highly efficient for transcription elongation but repressive to transcription-coupled DNA repair (TCR) .

  Research has shown that SPT5 is involved in multiple stages of gene expression beyond elongation, including pre-mRNA processing and splicing , demonstrating its multifunctional role in coordinating transcription with RNA processing.

• How does the domain architecture of SPT5 contribute to its functionality?

  SPT5 contains several functional domains that mediate specific interactions and functions:

  Domain	Location	Primary Function	Key Interactions
  NGN	N-terminal	Binds SPT4; key for pausing	Template DNA strand; RNA Pol II
  KOW1	Central	Mediates EC association	Upstream DNA
  KOW4-5	C-terminal	Extensive interactions with Pol II	Rpb4/7 subcomplex
  CTR	C-terminal	Phosphorylation site	P-TEFb kinase target

  The NGN domain contains a short helical motif critical for promoter-proximal pausing and is highly conserved in eukaryotes that encode NELF . The KOW4 domain facilitates pausing through its interaction with nascent RNA, while the KOW1 domain is required for DSIF association with the Pol II elongation complex . Deletion of the KOW4-5 domains decreases transcription elongation efficiency and derepresses transcription-coupled DNA repair .

• What is the evolutionary significance of SPT5 conservation?

  SPT5 stands out as the only known transcription factor conserved throughout all kingdoms of life, from bacteria to humans . This exceptional conservation underscores its fundamental importance in transcription mechanisms.

  In bacteria, the SPT5 homolog NusG shares sequence similarity with eukaryotic SPT5, particularly in regions that directly interact with RNA polymerase . The evolutionary relationship between bacterial NusG and eukaryotic SPT5 highlights the ancient origins of transcription elongation control mechanisms.

  The helical motif in the NGN domain of SPT5 that facilitates promoter-proximal pausing is notably present in eukaryotes that encode NELF but absent in organisms lacking promoter-proximal pausing, such as Saccharomyces cerevisiae and Caenorhabditis elegans . This pattern of conservation provides insight into the evolution of transcriptional regulation mechanisms across species.

Experimental Design and Methodological Approaches

• What are the recommended assays for studying SPT5-Pol II interactions in vitro?

  Several robust methodologies have proven effective for investigating SPT5-Pol II interactions:

  • Electrophoretic Mobility Shift Assays (EMSAs): These can assess the binding of recombinant SPT5 (or SPT4/5 complex) to purified RNA Pol II assembled on a tailed template with a G-less cassette. Typically, Cy5-labeled templates with 26-nucleotide-long transcripts are used to visualize the formation of elongation complexes .

  • Site-Specific Crosslinking: Incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive crosslinker, into specific positions of SPT5 allows precise mapping of interaction sites with RNA Pol II subunits. This technique has successfully identified interactions between SPT5 KOW4-5 domains and the Rpb4/7 subcomplex of Pol II .

  • Co-Immunoprecipitation: This approach has been used to demonstrate that SPT5 physically associates with RNA Pol II in vivo and can identify specific Pol II complexes containing SPT5 .

  • CUT&RUN (Cleavage Under Targets and Release Using Nuclease): This technique has been employed to study SPT5's genome-wide distribution and its role in transcription elongation. For DRB washout experiments to measure elongation rates, cells can be treated with 125 nM dTAGv-1 followed by 100 μM DRB, then collected at various timepoints after DRB removal .

• How can researchers assess the functional effects of SPT5 mutations?

  Multiple complementary approaches can be used to evaluate how SPT5 mutations affect its function:

  • Genetic Suppressor Analysis: Test if mutations in RNA Pol II can suppress phenotypes caused by SPT5 mutations. For example, rpb1 mutations have been shown to suppress the cold-sensitive phenotype of spt5 mutants in yeast, indicating a functional interaction between these factors .

  • Transcription Elongation Rate Measurements: Use DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) washout experiments combined with CUT&RUN or other genomic approaches to measure how SPT5 mutations affect transcription elongation rates. This has revealed that some SPT5 mutations can impact the timing of transcriptional pause release .

  • Northern Blot Analysis: Examine steady-state mRNA levels to assess transcription defects. In yeast, spt5 cold-sensitive mutations caused reduced levels of TPI1 and HIS4 mRNAs when cells were shifted to the nonpermissive temperature (13°C) .

  • Sensitivity to 6-Azauracil: Test growth on media containing 6-azauracil (6AU), as sensitivity to this compound is associated with transcription elongation defects. Many spt5 mutants and their genetic interactors display 6AU sensitivity .

• What approaches are effective for studying the role of SPT5 in RNA Polymerase I transcription?

  Several experimental strategies have proven useful for investigating SPT5's role in Pol I transcription:

  • Genetic Interaction Studies: Test interactions between spt5 mutations and mutations in Pol I subunits. For example, spt5 mutations suppress the cold-sensitive phenotype of rpa49Δ (encoding the A49 subunit of Pol I), suggesting that SPT5 plays a role in Pol I transcription .

  • rRNA Synthesis Rate Measurements: Quantify the synthesis rate of ribosomal RNA to assess how SPT5 mutations affect Pol I activity. This approach has revealed that deletion of SPT4 leads to increased rRNA synthesis rates per transcribing Pol I enzyme .

  • Pol I Occupancy Analysis: Use chromatin immunoprecipitation to measure Pol I occupancy on the rDNA. Combined with rRNA synthesis rate measurements, this can determine if SPT5 affects Pol I initiation, elongation, or both .

  • Electron Microscopic Analysis: This approach can provide direct visualization of Pol I transcription and has supported the model that SPT4/5 may contribute to pausing of Pol I early during transcription elongation but promotes elongation downstream of the pause sites .

Advanced Research Topics

• How does SPT5 contribute to promoter-proximal pausing and subsequent release?

  Promoter-proximal pausing is a critical regulatory step in metazoan gene transcription, and SPT5 plays a central role in this process:

  The SPT5-containing DSIF complex, along with Negative Elongation Factor (NELF), induces pausing of Pol II approximately 20-60 nucleotides downstream of the transcription start site. Recent research has identified specific domains of SPT5 that facilitate this pausing:

  • The NGN domain contains a short helical motif that promotes pausing through interaction with the non-transcribed DNA template strand .

  • The KOW4 domain interacts with the nascent RNA transcript to facilitate pausing .

  Pause release occurs when P-TEFb (positive transcription elongation factor b) phosphorylates the C-terminal region (CTR) of SPT5 and the C-terminal domain (CTD) of the largest Pol II subunit. This phosphorylation converts SPT5 from a pausing factor to an elongation-promoting factor .

  Recent findings suggest that SPT5 undergoes phase transition during transcriptional pause release. The Super Elongation Complex (SEC) induces SPT5 transition into "elongation droplets," and depletion of SEC increases SPT5 pausing clusters. Disease-associated SEC mutations impair the phase properties of these elongation droplets and disrupt transcription .

• What genetic evidence supports SPT5's role in transcription elongation?

  Multiple lines of genetic evidence firmly establish SPT5's function in transcription elongation:

  Conditional mutations in SPT5 can be suppressed in an allele-specific manner by mutations in the two largest subunits of RNA Pol II (Rpb1 and Rpb2). Notably, one of these RNA Pol II mutants (rpb1-221) is defective for transcription elongation, and the others cause phenotypes consistent with elongation defects .

  SPT5 mutants display several phenotypes associated with transcription elongation defects:

  • Sensitivity to 6-azauracil (6AU), a drug that depletes nucleotide pools and exacerbates elongation defects

  • Genetic interactions with known elongation factors

  • Reduced mRNA levels for some genes when shifted to nonpermissive temperature

  In Drosophila, mutations in SPT5 affect dosage compensation, which involves upregulation of transcription from the male X chromosome. Specifically, SPT5 mutations lower MSL (Male Specific Lethal) complex-dependent expression from reporter genes. SPT5 interacts directly with MSL1 in vitro and is required downstream of MSL complex recruitment, providing strong support for the elongation model of dosage compensation .

• How does SPT5 coordinate transcription with pre-mRNA processing?

  SPT5 serves as a critical coordinator between transcription elongation and various RNA processing events:

  Studies in yeast have demonstrated that mutations in SPT4 and SPT5 display genetic interactions with mutations in capping enzyme genes. Additionally, these mutations lead to accumulation of unspliced pre-mRNA, indicating a role in pre-mRNA splicing .

  The human and Schizosaccharomyces pombe SPT5 proteins directly interact with the capping enzyme, suggesting that SPT5 helps recruit RNA processing factors to the nascent transcript .

  SPT5 interacts extensively with the Pol II CTD, which serves as a platform for the recruitment of RNA processing factors. This interaction may help coordinate transcription elongation with co-transcriptional RNA processing .

  Through coimmunoprecipitation analyses, research has shown that SPT5 participates in at least three different protein complexes with Pol II, providing further evidence of its role in coordinating various aspects of gene expression .

• What is the role of SPT5 in human genetic disorders?

  Recent research has identified loss-of-function variants in SUPT5H (the human gene encoding SPT5) as modifying factors in beta-thalassemia:

  Carriers of SUPT5H loss-of-function variants display a beta-thalassemia-like phenotype characterized by elevated levels of HbA2, even in the absence of mutations in the HBB (beta-globin) gene . This suggests that SPT5 plays a role in regulating beta-globin gene expression.

  In individuals who are double heterozygotes (carrying both an SUPT5H variant and an HBB variant), a mild beta-thalassemia intermedia phenotype is observed, with more severe hematological parameters than seen in typical beta-thalassemia carriers .

  Group	Hb (g/L)	MCV (fL)	MCH (pg)	HbA2 (%)
  Normal	120-170	80-100	27-32	2.0-3.5
  β-thalassemia carriers	100-140	60-75	18-23	3.5-7.0
  SUPT5H carriers	100-140	70-85	22-27	3.5-7.0
  SUPT5H/HBB double heterozygotes	<120	<70	<20	8.0-11.0

  Mechanistic studies involving CRISPR-Cas9-mediated perturbation of SUPT5H in human hematopoietic stem and progenitor cells have shown that reduced SPT5 levels affect enhancer transcription at the beta-globin locus control region (LCR), suggesting a mechanism for how SUPT5H variants impact beta-globin expression .

Methodological Techniques for SPT5 Research

• What approaches are effective for studying SPT5-mediated phase transitions?

  Studying the recently discovered phase transition properties of SPT5 requires specialized techniques:

  • Live Cell Imaging: Fluorescently tagged SPT5 can be used to visualize the formation of SPT5 clusters or "elongation droplets" in living cells. This approach has shown that SPT5 is prone to form clusters and that the super elongation complex (SEC) induces SPT5 transition into elongation droplets .

  • Fluorescence Recovery After Photobleaching (FRAP): This technique can assess the dynamics of SPT5 within phase-separated droplets, providing insights into the material properties of these structures and how they might be regulated.

  • Truncation and Mutation Analysis: The disordered domain in SPT5 is required for pause release and gene activation. Creating SPT5 variants with mutations or deletions in this domain can help identify the regions essential for phase transition .

  • SEC Depletion and Mutation Studies: Depletion of the super elongation complex increases SPT5 pausing clusters, and disease-associated SEC mutations impair the phase properties of elongation droplets. These approaches can reveal the molecular mechanisms regulating SPT5 phase transitions .

• How can researchers identify and characterize novel SPT5-interacting proteins?

  Multiple complementary approaches can be used to discover and validate SPT5 interaction partners:

  • Affinity Purification Coupled with Mass Spectrometry: This approach has successfully identified numerous proteins that copurify with SPT5 from yeast extracts. For example, this method identified that Spt5 associates with Pol II and general elongation factors TFIIF and TFIIS, as well as with Spt6, Cdc68, and Pob3 .

  • Coimmunoprecipitation: This technique can validate potential interactions identified by mass spectrometry and can be used to map the specific complexes containing SPT5. Studies have shown that SPT5 participates in at least three different protein complexes with Pol II .

  • Yeast Two-Hybrid Screens: This genetic approach can identify direct protein-protein interactions involving SPT5 or its domains.

  • Genetic Interaction Screens: Identifying mutations in other genes that suppress or enhance phenotypes caused by SPT5 mutations can reveal functional interactions. For example, an unbiased forward genetic screen in Drosophila identified a connection between SPT5 and the MSL complex involved in dosage compensation .

• What are the optimal approaches for studying the effects of SPT5 on RNA polymerase dynamics?

  Several advanced techniques can provide insights into how SPT5 affects the dynamics of RNA polymerase:

  • DRB Washout Experiments: Treating cells with DRB or flavopiridol blocks P-TEFb phosphorylation of Pol II CTD Ser2 and SPT5, trapping new polymerase at the transcription start site. After drug removal, the rate at which polymerase progresses along genes can be measured to determine elongation rates. This approach has been used to show that SPT5 phosphorylation regulates Pol II elongation rates both positively and negatively .

  • CUT&RUN and ChIP-seq: These genomic approaches can map the distribution of SPT5 and Pol II genome-wide. After DRB washout, these techniques can track the wave of transcribing polymerase as it progresses through genes .

  • Structural Studies: Cryo-electron microscopy of transcription elongation complexes containing SPT5 has revealed that SPT5 interacts with the clamp, protrusion, and wall domains of Pol II, potentially locking the clamp in a closed conformation that encloses the DNA being transcribed .

  • Transcription Assays with Purified Components: In vitro transcription systems using purified Pol II, SPT4/5, and other factors allow detailed mechanistic studies of how SPT5 affects transcription elongation rates, pausing, and arrest .

• How should researchers approach the design of SPT5 mutations for functional studies?

  Strategic design of SPT5 mutations can provide valuable insights into its structure-function relationships:

  • Charge Reversal Mutations: Reversing the charges of basic nucleic acid-interacting residues of SPT5 has been effective in disrupting specific interactions. For example, mutating six conserved positively charged residues in the KOW1 domain to aspartic acid disrupts its interaction with upstream DNA .

  • Domain Deletion/Truncation: Deleting specific domains, such as the KOW4-5 domains that interact with Rpb4/7, can reveal their functional importance. Such deletions have been shown to decrease transcription elongation and deregulate transcription-coupled DNA repair .

  • Evolutionary Analysis-Based Mutations: The short helical motif in the NGN domain that is critical for pausing is conserved in eukaryotes that encode NELF but absent in organisms lacking promoter-proximal pausing. Replacing this motif with homologous sequences from Saccharomyces cerevisiae and Caenorhabditis elegans results in a male-specific dominant negative effect in Drosophila .

  • Disease-Associated Variants: Studying naturally occurring SUPT5H variants identified in human genetic disorders, such as those found in individuals with beta-thalassemia-like phenotypes, can provide insights into SPT5 function in specific biological contexts .