Stable Elongation Research Articles

Mechanical and energy absorption behaviors of 3D printed continuous carbon/Kevlar hybrid thread reinforced PLA composites

Over the last few years, three-dimensional printing(3DP) technology has evolved to incorporate composite materials. However, using material extrusion (ME) technology, it is still difficult to improve the stiffness with stable elongation and energy absorption of continuous fiber reinforced composites at the same time. This study aimed to 3D print hybrid threads by merging several types of continuous Kevlar and Carbon fibers. Which were created using material extrusion 3D printing to accomplish cooperative ductility and brittleness augmentation. To analyze the mechanical performance and reveal the failure and deformation mechanisms, tension tests, three-point bending tests, and drop hammer impact tests were performed. The proposed hybrids have substantially greater flexural modulus, flexural strength, tension modulus, and tension strengths with absorbed energies than standard single type fiber composites. Crack resistance during impact test of hybrid composites was enhanced by over 91% compared to carbon fiber non-hybrid composites. SEM images of hybrid specimens provided an insight of the failure mechanisms, resulting in stable hybrid composites. Positive hybrid effect further demonstrates its great energy absorption capacity.

Effect of plant tannin and glycerol on thermoplastic starch: Mechanical, structural, antimicrobial and biodegradable properties

Thermoplastic starch is a good candidate for preparing biobased plastics. However, certain deficiencies in the ingredient, such as its poor mechanical strength and lack of antibacterial activity, need to be strengthened. In this work, a new method of producing TPS via extrusion and injection at a large scale was explored using tannin and glycerol simultaneously. The effect of the tannin content on the performance of TPS was investigated. The results showed that the synergistic effect of a small amount of tannin (2.5–7.5 wt%) and glycerol increased the tensile strength and maintained a stable elongation at break, and tannin aggregation occurred in TPS at higher tannin contents, which corresponded to increased tensile and flexural strengths. 15T-TPS has optimal mechanical performance with maximal relative crystallinity. The introduced tannin in TPS effectively enhanced the thermal stability but slightly reduced the biodegradability in soil and endowed TPS with antibacterial activity. Therefore, TPS containing tannins has the potential to be used in biomedical, food packaging and agricultural areas.

Unique Biochemical Properties of Eukaryotic RNA polymerases I, II, and III

Eukaryotes express at least three nuclear, DNA‐dependent RNA polymerases (Pols I, II, and III) responsible for synthesizing all genome‐encoded RNA. Based on the Pols’ cellular locations, target loci, and transcriptional demands, it is reasonable to hypothesize they have diverged enzymatically over the course of evolution. Since the discovery of the Pols over 50 years ago, the Pols have been largely investigated separately and under disparate experimental conditions, making direct comparisons of the Pols unjustifiable. To directly compare the Pols under identical experimental conditions, we utilized our promoter‐independent in vitro transcription assays. We use a rapid mixing instrument, the chemical quenched‐flow, to analyze single‐ and multi‐nucleotide addition catalyzed by the Pols. We find that Pol I incorporates a single AMP faster than Pols II and III. Interestingly, Pol III incorporates multiple nucleotides faster than Pol I on average. Pol II is consistently the slowest Pol over single‐ and multi‐nucleotide incorporation events. Outside of elongation kinetics, we find additional distinct biochemical properties. Pol II forms the most stable elongation complex of the Pols and is the least error prone Pol. Taken together, our results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.

Fiber-level numerical simulation of biaxial braids for mesoscopic morphology prediction validated by X-ray computed tomography scan

This paper proposes a modeling methodology to predict the 3D and internal geometry of biaxial braids. Inspired by the digital element approach, braided yarns are modeled as a bundle of virtual fibers. Here we adopt truss elements to a virtual fiber with actual material properties instead of beam elements that have limitations due to the beam flexural rigidity. The mesoscopic morphology prediction of two common braid patterns of the diamond (1/1) and regular (2/2) are validated by a comprehensive quantitative comparison with X-ray micro-computed tomography (CT) scans of braided carbon fibers. We find that the fiber-level frictional behavior is able to explain the jammed state of braids wherein the frictional dissipation energy quickly grows, while the braid has a stable elongation, diameter, and braid angle. Parametric studies illustrate how the increase in the coefficient of friction affects the yarn cross-sectional shape, whereas it has an insignificant effect on the crimp and jammed state of braids. Models also reveal that changing a wide range of the fiber modulus of elasticity hardly impacts the mesoscopic morphology and crimp of the braids.

In vitro evaluation of the ZX11 magnesium alloy as potential bone plate: Degradability and mechanical integrity

Considering the excellent biocompatibility of magnesium (Mg) alloys and their better mechanical properties compared to polymer materials, a wrought MgZnCa alloy with low contents of Zn (0.7 wt%) and Ca (0.6 wt%) (ZX11) was developed by twin roll casting (TRC) technology as potential biodegradable bone plates. The degradability and cell response of the ZX11 alloy were evaluated in vitro, as well as the mechanical integrity according to tensile tests after immersion. The results revealed a slightly higher degradation rate for the rolled ZX11, in comparison to that of the annealed one. It was mainly caused by the deformation twins and residual strain stored in the rolled alloy, which also seemed to promote localized degradation, thereby leading to a relatively fast deterioration in mechanical properties, especially the fracture strain/elongation. In contrast, after the annealing treatment, the alloy showed relatively lower strength, yet a lower degradation rate and quite stable elongation during the initial weeks of immersion were observed. More importantly, the ZX11 alloy, regardless of the annealing treatment, showed good in vitro cytocomopatibility regarding human primary osteoblasts. The assessment indicates the rolled alloy as a good choice for implantation sites where relatively high mechanical strength is needed during the early implantation, while the annealed alloy is a potential candidate for the sites which demand stable mechanical integrity during service. Statement of SignificanceThe development of magnesium alloys as bone implants demands low degradation rate to gain not only a slow hydrogen evolution, but also a stable mechanical integrity during service. The present study develops a micro-alloyed MgZnCa alloy via twin roll casting (TRC) technology. It exhibited limited cytotoxicity, fairly low degradation rate and comparable strength to the reported Mg-1Zn-5Ca alloy which has been used as bone screws in clinical trials, indicating the great potential application as biodegradable bone implants. Furthermore, it showed good mechanical integrity during immersion to support the defect healing. Our results can aid other researchers to evaluate the mechanical integrity of biodegradable materials and to pay more attention to the effect of degradation behaviour on mechanical integrity of materials.

Response of retained austenite to quenching temperature in a novel low density Fe-Mn-Al-C steel processed by hot rolling-air cooling followed by non-isothermal partitioning

The quenching temperatures below Ms are difficult to uniformly control during large-scale processing of quenched and partitioned (Q&P) steels, which results in significant variation in tensile properties for the entire coil. The present study is aimed at obtaining a stable elongation for a wide range of quenching temperatures in hot rolled Q&P steels through microstructural design. Instead of cold rolling and annealing processes, a novel process involving hot rolling and air cooling followed by cooling in furnace was applied to a newly designed 0.25C-3Mn-2Al (wt.%) steel. The results indicated that a stable amount of retained austenite (RA) ∼ 19–23% was obtained during air-cooling to finish temperatures in the range of ∼280–360 °C, resulting in no loss in elongation at low quenching temperatures. The RA that was promoted by the transfer of carbon from ferrite/bainite reached up to 1.346 μm, whereas the RA embedded in martensitic laths was ∼40 nm. The two types of RA provided continuous TRIP effect during tensile deformation and the ultra-fine martensite lath of ∼100 nm ensured tensile strength greater than 1091 MPa. Finally, a stable total elongation of ∼19.2–20.5% in combination with tensile strength of ∼1091–1196 MPa was obtained over a wide quenching temperature range of 80 °C, which broadened the processing window for the production of hot rolled Q&P steels. In addition, the isothermal transformation during the initial partitioning stage was the underlying reason to obtain stable amount of RA. The CCE (Constrained Carbon Equilibrium) model combined with isothermal transformation was used to quantitatively calculate the volume fraction of RA.

Effect of film properties on formability of aluminum pouch for secondary battery

The present study was conducted to investigate the effect of film properties on the formability of aluminum pouches for secondary batteries. Among the physical properties of the film, the effects of the friction coefficient and the elongation rate on the formability were investigated. The purpose of this study is to draw the improvement of formability according to the physical properties of the films for constructing the aluminum pouch on the assumption that the Friction resistance between the mold and material in the forming process and the Tensile resistance generated when the material is deformed by the punch will affect the formability. The results showed that the formability was better when the friction coefficient was lower. Specially, the formability is improved more in the sample that had the nylon film with a reduced friction coefficient than in the sample with the CPP film that had a reduced friction coefficient. This indicates that the limit of bending deformation was extended as the friction coefficient of the nylon film get lower. In addition, we found that the formability depends on the elongation property. The formability is improved when the nylon film with isotropic property and a CPP film with stable elongation property were applied. In a sample NY.2/CPP.2 manufactured by using an isotropic nylon and a stably elongated CPP film, the formability has maximum forming depth of 5.5 mm. This result verified that materials that have low friction coefficient, isotropy and a stable elongation property may be applied to improve the formability.

Distal CpG islands can serve as alternative promoters to transcribe genes with silenced proximal promoters.

DNA methylation at the promoter of a gene is presumed to render it silent, yet a sizable fraction of genes with methylated proximal promoters exhibit elevated expression. Here, we show, through extensive analysis of the methylome and transcriptome in 34 tissues, that in many such cases, transcription is initiated by a distal upstream CpG island (CGI) located several kilobases away that functions as an alternative promoter. Specifically, such genes are expressed precisely when the neighboring CGI is unmethylated but remain silenced otherwise. Based on CAGE and Pol II localization data, we found strong evidence of transcription initiation at the upstream CGI and a lack thereof at the methylated proximal promoter itself. Consistent with their alternative promoter activity, CGI-initiated transcripts are associated with signals of stable elongation and splicing that extend into the gene body, as evidenced by tissue-specific RNA-seq and other DNA-encoded splice signals. Furthermore, based on both inter- and intra-species analyses, such CGIs were found to be under greater purifying selection relative to CGIs upstream of silenced genes. Overall, our study describes a hitherto unreported conserved mechanism of transcription of genes with methylated proximal promoters in a tissue-specific fashion. Importantly, this phenomenon explains the aberrant expression patterns of some cancer driver genes, potentially due to aberrant hypomethylation of distal CGIs, despite methylation at proximal promoters.

Hydrophobic and charged residues in the C-terminal arm of hepatitis C virus RNA-dependent RNA polymerase regulate initiation and elongation.

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

Transcription regulation during stable elongation by a reversible halt of RNA polymerase II.

Regulation of RNA polymerase II (RNAPII) during transcription is essential for controlling gene expression. Here we report that the transcriptional activity of RNAPII at the Balbiani ring 2.1 gene could be halted during stable elongation in salivary gland cells of Chironomus tentans larvae for extended time periods in a regulated manner. The transcription halt was triggered by heat shock and affected all RNAPII independently of their position in the gene. During the halt, incomplete transcripts and RNAPII remained at the transcription site, the phosphorylation state of RNAPII was unaltered, and the transcription bubbles remained open. The transcription of halted transcripts was resumed upon relief of the heat shock. The observed mechanism allows cells to interrupt transcription for extended time periods and rapidly reactivate it without the need to reinitiate transcription of the complete gene. Our results suggest a so-far-unknown level of transcriptional control in eukaryotic cells.

The Mechanics of Initial Transcription: The RNA-DNA Hybrid Pushes on the Protein, and the Protein Pushes Back!

RNA polymerases must couple the energetics of nucleotide addition to drive timed release of strong promoter contacts. RNA polymerases also undergo substantial structural changes in transitioning from sequence-specific initial transcription to stable elongation. Initially transcribing complexes are characteristically unstable, yielding short abortive products, but polymerase mutations have been isolated that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. using fluorescence probes of structural changes, we demonstrate that the low-abortive P266L mutant T7 RNA polymerase also transitions to elongation at longer lengths of RNA. We propose that both properties derive from a weakening of the initial barrier to hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to drive promoter release. While the hybrid pushes on this protein element, the protein in turn pushes back on the hybrid, leading to the observed instability during initial transcription. Parallel biochemical experiments on bacterial RNA polymerase show that the sigma region 3.2 linker (analogous to the eukaryotic RNA polymerase “B-finger”) functions in a very similar manner. Thus the mechanical “pushing” of the RNA-DNA hybrid against the protein is met by a reciprocal, and destabilizing, pushing of the protein against the hybrid.

New Insights into the Mechanism of Initial Transcription

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

Insights into Abortive Cycling during Initial Transcription by T7 RNA Polymerase

RNA polymerases are characterized by conflicting requirements during initial transcription and elongation. In the initial phase, an RNA polymerase must recognize a specific promoter sequence, melt the DNA at the start site, initiate de novo synthesis of a dimer RNA, and extend that RNA in a hybrid that is thermodynamically unstable to collapse of the melted bubble. Hybrid growth in turn ultimately drives promoter release and a structural transition that enables topologically locked RNA entry into a stabilizing exit channel, establishing a stable elongation complex.Perhaps not surprisingly, short RNAs are not stable during initial transcription, leading to their abortive release. But hybrid stability is only part of the story. In T7 RNA polymerase, a single point mutation (P266L) in the protein, distant from the hybrid, leads to a dramatic reduction in abortive release on most promoters. Current models suggest that the mutant releases promoter contacts more readily, transitioning to a stable elongation complex sooner.New results reveal the opposite behavior: P266L releases promoter contacts later than wild type, suggesting an overall increase in stability of the initially transcribing complex. Based on kinetic studies, we present a testable model to explain how growth of the hybrid serves to drive a timed transition to elongation.

Role of DNA Bubble Collapse in Initiation and the Transition to Elongation-A Common Mechanism in all RNA Polymerases

The well-studied RNA polymerases fall into two distinct classes: the “single subunit” family represented by bacteriophage T7, mitochondrial, and chloroplast RNA polymerases and the “multi-subunit” bacterial and eukaryotic enzyme family. All RNA polymerases are characterized by a relatively unstable initial phase (abortive cycling) and by a significant structural rearrangement associated with the transition to stable elongation. Our previous studies with the single subunit T7 RNA polymerase have established a key role for downstream DNA bubble collapse in the instability of halted initiating complexes. We now extend these studies to the initially transcribing complexes of sigma70-E. Coli RNA polymerase transcribing on variants of the T5 N25 promoter. Specifically, we have studied the role of downstream DNA bubble collapse in initially transcribing complexes by designing partially single stranded DNA constructs with the nontemplate absent at the downstream edge of the transcription bubble. As in our earlier studies, the current results show that collapse of the downstream end of the transcription bubble contributes to instability of a halted complex. This increases turnover and hence the amount of abortive transcripts. Transcription to elongation on these constructs show 18-23mer RNA products. We propose that absence of the collapse of the upstream end of the transcription bubble impairs proper displacement of the 5’end of RNA into the exit channel, leading to unstable complexes. This parallels our earlier model in the T7 system in which upstream bubble collapse (−4 to +2) facilitates displacement of the RNA from the hybrid (impairment of collapse leads to release of 11-13mer RNAs) enabling the transition to elongation.

Structural basis of initial RNA polymerase II transcription

During transcription initiation by RNA polymerase (Pol) II, a transient open promoter complex (OC) is converted to an initially transcribing complex (ITC) containing short RNAs, and to a stable elongation complex (EC). We report structures of a Pol II-DNA complex mimicking part of the OC, and of complexes representing minimal ITCs with 2, 4, 5, 6, and 7 nucleotide (nt) RNAs, with and without a non-hydrolyzable nucleoside triphosphate (NTP) in the insertion site +1. The partial OC structure reveals that Pol II positions the melted template strand opposite the active site. The ITC-mimicking structures show that two invariant lysine residues anchor the 3'-proximal phosphate of short RNAs. Short DNA-RNA hybrids adopt a tilted conformation that excludes the +1 template nt from the active site. NTP binding induces complete DNA translocation and the standard hybrid conformation. Conserved NTP contacts indicate a universal mechanism of NTP selection. The essential residue Q1078 in the closed trigger loop binds the NTP 2'-OH group, explaining how the trigger loop couples catalysis to NTP selection, suppressing dNTP binding and DNA synthesis.

The Transcriptional Repressor CcpN from Bacillus subtilis Uses Different Repression Mechanisms at Different Promoters

CcpN, a transcriptional repressor from Bacillus subtilis that is responsible for the carbon catabolite repression of three genes, has been characterized in detail in the past 4 years. However, nothing is known about the actual repression mechanism as yet. Here, we present a detailed study on how CcpN exerts its repression effect at its three known target promoters of the genes sr1, pckA, and gapB. Using gel shift assays under non-repressive and repressive conditions, we showed that CcpN and RNA polymerase can bind simultaneously and that CcpN does not prevent RNA polymerase (RNAP) binding to the promoter. Furthermore, we investigated the effect of CcpN on open complex formation and demonstrate that CcpN also does not act at this step of transcription initiation at the sr1 and pckA and presumably at the gapB promoter. Investigation of abortive transcript synthesis revealed that CcpN acts differently at the three promoters: At the sr1 and pckA promoter, promoter clearance is impeded by CcpN, whereas synthesis of abortive transcripts is repressed at the gapB promoter. Eventually, we demonstrated with Far Western blots and co-elution experiments that CcpN is able to interact with the RNAP alpha-subunit, which completes the picture of the requirements for the repressive action of CcpN. On the basis of the presented results, we propose a new working model for CcpN action.

Probing DNA mechanical characteristics by dielectrophoresis

Microfabricated electrode systems have recently become useful for immobilization and stretching biopolymers to precise locations in microfluidic devices. In this paper we discuss the modeling, fabrication and characterization of such a platform to investigate the elongation and orientation of different-sized deoxyribose nucleic acid (DNA) molecules, tethered onto aluminum electrodes, by a distributed force applied along the DNA backbone under the influence of high frequency non-uniform ac electric fields. The DNA molecules are elongated from a random coil into an extended conformation and orientated along the electric field lines as a result of the forces acting on the molecules during the application of the ac electric fields. Stable elongation is observed in the frequency range 0.1–1 MHz, with field strengths of 0.3–1.9 MV/m. Maximum elongation for two different DNA fragments, irrespective of size, is found for frequencies about 100 kHz. With a model that incorporates dielectrophoresis directly, we show that electric field experimental results can be used for detecting some characteristics of the DNA elasticity which manifest themselves clearly at higher extensions but cannot be observed at lower ones. The entropic elasticity and highly extensibility of DNA are all closely related to this account. By the elastic DNA model presented and by considering the base–stacking interactions between DNA adjacent nucleotide base pairs, we find an underlying scaling relation between the DNA extension and the applied voltage of the form 〈 δ 〉 ∼ V p − p 1 − 1.15 , which emphasizes the significance of the electric fields.

Kinetic Investigation of Escherichia coli RNA Polymerase Mutants That Influence Nucleotide Discrimination and Transcription Fidelity

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.

Cross-linking of Promoter DNA to T7 RNA Polymerase Does Not Prevent Formation of a Stable Elongation Complex

T7 RNA polymerase recognizes a small promoter, binds DNA, and begins the process of transcription by synthesizing short RNA products without releasing promoter contacts. To determine whether the promoter contact must be released to make longer RNA products and at what position the promoter must be released, a mutant RNA polymerase was designed that allows cross-linking to a modified promoter via a covalent disulfide bond. The modifications individually have no measurable effect on transcription. Under oxidizing conditions that produce the protein-DNA cross-link, the complex is able to synthesize short RNA products, strongly supporting a model in which promoter contacts are not lost on translocation through at least position +6. However, cross-linked complexes are impaired in promoter escape in that only about one in four can escape to make full-length RNA. The remainder release 12- and 13-mer RNA transcripts, suggesting an increased energetic barrier in the transition from an initial transcribing complex to a fully competent elongation complex. The results are discussed in the context of a model in which promoter release helps drive initial collapse of the upstream edge of the bubble, which, in turn, drives initial displacement of the 5'-end of the RNA.

A Conserved Zinc Binding Domain in the Largest Subunit of DNA-dependent RNA Polymerase Modulates Intrinsic Transcription Termination and Antitermination but does not Stabilize the Elongation Complex

An evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of DNA-dependent RNA polymerases from bacteria, archaea, and eukaryotes. In bacterial RNA polymerase, this motif, the zinc binding domain, has been implicated in protein-DNA interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. Here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product RNA located upstream of the catalytic center and the RNA-DNA hybrid, a view consistent with structural studies of the elongation complex. We engineered mutations that alter or remove the zinc binding domain of Escherichia coli RNA polymerase. Several mutants, including one that lacked all four zinc ligands and another that lacked the entire domain, produced enzymes that were active in vitro and formed stable elongation complexes. However, they were defective in two functions that require interaction of polymerase with product RNA. First, they terminated less efficiently than the wild-type at intrinsic transcription terminators. Second, enzymes lacking the tip of the zinc binding domain or the zinc ligands did not antiterminate in response to an intrinsic antiterminator encoded by the put site of phage HK022. Termination, but not antitermination, was restored by the bacterial termination factor NusA. Surprisingly, a mutant that lacks the entire zinc binding domain regained a partial response to put. To account for this we suggest that put RNA interacts with an additional site in the elongation complex to mediate antitermination, and that this site is occluded by the wild-type zinc binding domain.