Bacterial Proteins Required for Replication of Phage Qa Ribonucleic Acid PURIFICATIOn’ ASD PROPERTIES OF HOST FACTOR

SUMMARY A bacterial protein (host factor I) required for the replication of bacteriophage Q/3 RNA in vitro has been purified from uninfected Escherichia coli to apparent homogeneity. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the purified protein migrates as a single band with an apparent molecular weight of 12,500. The molecular weight of the active protein is approximately 75,000, suggesting a subunit structure composed of six polypeptide chains. RNA synthesis catalyzed by the Qfl RNA polymerase with Qfi RNA as template is completely dependent upon the presence of the purified protein. The concentration of required for the maximum rate of synthesis varies according to the amount of Q/3 but is independent of enzyme concentration. The stoichi-ometry of the 1 molecule of factor is required per molecule several reovirus

A bacterial protein (host factor I) required for the replication of bacteriophage Q/3 RNA in vitro has been purified from uninfected Escherichia coli to apparent homogeneity. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the purified protein migrates as a single band with an apparent molecular weight of 12,500.
The molecular weight of the active protein is approximately 75,000, suggesting a subunit structure composed of six polypeptide chains. RNA synthesis catalyzed by the Qfl RNA polymerase with Qfi RNA as template is completely dependent upon the presence of the purified protein.
The concentration of factor required for the maximum rate of synthesis varies according to the amount of Q/3 RNA added, but is independent of enzyme concentration. The stoichiometry of the reaction suggests that 1 molecule of factor is required per molecule of RNA.
Binding of factor to several different types of single stranded RNA was demonstrated by zonal centrifugation; binding was not detected with double stranded reovirus RNA or with double or single stranded DNA.
Studies on the role of factor in the reaction suggest that this host protein acts as a positive control element required at some step prior to the initiation of synthesis with QP RNA template.
It is becoming increasingly apparent that the control of gene replication aud transcription depends on a large number of protein factors.
Well characterized examples are the h and lac reprcssors, the X N-gene product, the ~7 subunit of RNh polymerase, the p factor, the T4 gene 32 product, and a cyclic atlellosine 3', 5'.monophosphate-binding protein. Protein factors also play a role in the control of the replication of bacteriophage RNA. Synthesis of QP RNA in z&o requires, in addition to the Qp RNA polymcrase, two proteins which call be isolated from either infected or uninfected Escherichin coli (1,2). These proteins, host factors I and II (HFI and IIF,,), al?pear to determine the specificity of the reaction, rather thall polymerization per se, since they are required only with Q@ RX.1 template; RNA synthesis with other templates, most notably the Qp complementary strand RNB, proceeds normally in the absence of either factor (3).
Several other host proteins hare been implicated in the in vitro replication of &a RNA.
Highly purified fractions of the llolymerase enzyme contain one I)hage-coded and three host peptides (4,5). It is assumed that all four peptides are necessary for polymerase activity, although the reconstitutiou of active enzyme from individual components has not been achier-cd.
This report describes the purification and some of the propcrties of host factor I. This factor is found to be an RNA\-binding protein which can be distinguished from other E. coli l)roteins known to be involved in transcription or translation.
1Waterials &S RNA Polymerase-Qfi RNA polymerase was purified and analyzed as previously described (6,7). One unit of enzyme is that amount catalyzing the incorporation of 1 nmole of GhIP per 20 min at 37" in a reaction mixture containing QS RNA as template and an excess of HF1 and HFI1 (6). When analyzed by polyacrylamide gel electrophoresis in the presence of SDS1 these enzyme preparations were found to contain only the four polypeptides which have been reported by others to be associated with purified enzyme (4,5), presumably as enzyme subunits.
Host Factor IZ-HF,I was purified by either of t\\-o methods. The initial studies described here utilized a partially purified preparation (hydroxylapatitc fraction) fret of IIFl and enzyme activity, prepared from phage Q&infected E. coli Q13 as previously described (2). ;I!lore recently a highly purified preparation was obtained from uninfected B. coli Q13 as described by Kuo (8). 1 The abbreviation used is: ST)S, sodium dodccyl slllfnlc.
Issue of February 10, 1972 Franxe de Fernanclex, Hayward, and August 525 Nucleic Acids-The RNA of Q/3, f2, and R23 coliphages was prepared by phenol extraction and ethanol precipitation (6). E. coli rRNA was prepared by phenol extraction of ribosomes isolated by high speed centrifugation (9). Reovirus RNA was isolated from purified virus (10) provided by Dr. A. R. Bellamy. After phenol extraction in the presence of 1% SDS the RNA was further purified by zonal centrifugation.
Fractions sedimenting at approximately 10 S were pooled, concentrated by ethanol precipitation, and dissolved in 50 mM Tris-acetate buffer, pH 7.7, and 2 mM EDTA.
This RNA showed a sharp hyperchromic thermal transition at 77" in 15 m&x sodium citrate-150 mM sodium chloride suggesting that at least 95yc was double stranded.2 fd DNA, prepared according to the method of Marvin and Schaller (II), was a gift of Dr. Paul Sadowsky; X CIgr,7 DNiZ, provided by Dr. David Axelrod, was prepared by a slight modification of the method of Baldwin et nl. (12) ; PBSX DNA, purified as described by Okamoto et cd. (13), was a gift of Dr. W. Huang.
Chemicals-All chemicals were purchased from standard sources.
[+P]GTP was prepared as previously described (14) with the use of GTP purified as described by Hurlbert and Furlong (15)  When a more sensitive method was required the protein was hydrolyzed by incubation in 20 yc NaOH for 20 min at 110", and the amino acid concentration was measured with ninhydrin by the method of Hill and Delaney (17). L-Leucine was used as standard. Samples containing ammonium sulfate were first dialyzed extensively against 25 mM NaCl and 4 rnnl 2-mercaptocthanol.
Amino Acid Analysis-The factor preparations (glycerol gradient fractions) were dialyzed for 7 hours against 25 mM NaCl and 4 rnhr 2-mercaptocthanol in order to remove ammonium sulfate. They were then adjusted to 6 PI' HCI and 0.5R 2-mercaptoetha-no1 and hydrolyzed in evacuated sealed tubes at 100" for 22 hours. The amino acid analysis was performed in a Spinco 1201~ analyzer equipped with 6.6-mm cells and a high sensit'ivity recorder.

Carbohydrate
Analysis-Samples (20 to 30 Kg of protein) were analyzed by gas-liquid chromatography according to the method of Albersheim et al. (18), with inositol as an internal standard.
Protein bands were stained at room temperature wit,h 0.05yc Coomassie brilliant blue in 12.5yc trichloroacetic acid for at least 12 hours and destained with 70/O acetic acid. The molecular weight of HFr was estimated as described by Shapiro et al. (20), by comparing its mobility with that of proteins of known molecular weight.
Assay of Host Factor I-Tkle assay measures the incorporation of [~*C]AMP into acid-insoluble material in the presence of Qp RNA, the Q@ RNA polymerase, and HFrr.
With the purified Qp RNA polymerase and the partially purified or highly purified HF,,, RNA synthesis with Qp RNA template lvas completely de-2 We are indebted to Dr. P. Silverman for analysis of reovirus RNA. pendent on added HFr (2). The assay mixture (0.1 ml) contained 100 mM Tris-HCl buffer, pH 7.6, 10 mM MgC12, 4 mM 2mercaptoethanol, 1 mM each of UTP, GTP, CTP, and [14C]ATP, 1 pmole of QP RNA, HFrI in excess (1 to 2 pg of protein of the purified fraction), 1.0 unit of the QP RNA poly-merase, and an appropriate amount of HF,.
After incubation for 20 min at 37" the reaction was terminated by adding 5% trichloroacetic acid cont'aining 20 mM PI';, and the acid-insoluble material was collected and washed on membrane filters as previously described (6). Reaction mixtures lacking HFr served as blanks.
The activity was routinely measured at several protein concentrations to establish a linear relationship between protein concentration and nucleotide incorporation (0.1 to 0.5 unit of HF, with 1 unit of polymerase). One unit of HFr is calculated from this linear range as that amount corresponding to the incorporation of 1 nmole of AMP under these standard conditions.
Association of Host Factor I with Polynucleotides-The binding of HFr to nucleic acids was carried out in a reaction mixture con taining 100 mM Tris-HCl buffer, pH 7.6, 10 mM MgCl*, 50 mM KCl, 4 rnl\i 2-mercaptoethanol, 30 to 60 pg of polynucleotide, and approxirnately 25 units of HFr in a total volume of 0.1 ml. After mixing at O-4" the sample was layered on a solution containing a linear gradient of 5 to 307; glycerol, 100 mM Tris-I-TCl buffer, pH 7.8, 10 MM MgC12, 50 mM KCl, and 4 m&I 2-mercaptoethanol and centrifuged in a Spinco SW 65 rotor as indicated in the legends. Fractions (0.25 ml) were collected from the bottom of the tube and analyzed for absorbance at 260 nm and for HF, activily. Pyrophosphate Exchange Reaction-The reaction mixture (0.25 ml) contained 100 mM Tris-HCl, pH 7.8, 10 rnbr lIgCIZ, 4 mM 2mercaptoethanol, 0.8 mM AT!?, UTP, GTP, and CTP, 1.5 mM Na32PPi (specific activity 7.4 x lo6 cpm per pmole), 1.3 pmoles of QP RNA, 2 units of the Qfl RNA polymerasc, 2 ,ug of HFr, (hydroxylapatite fraction), and HFr as indicated. After incubation at 37" for 20 min the reaction was terminated by the addition of approximately 3 ml of IO?; trichloroacetic acid containing 0.2 M NaPPi.
Norit (0.1 ml of a 307; solution) was added with occasional mixing and after 10 min the suspension was filtered through a Millipore filter and washed 4 times with approximately 3 ml of 0.2 M NaPPi.
The filters were glued to planchets Tvith the Norit facing downward and dried. The radioactivity was measured in a gas flow counter. After incubation for 20 min at 37" the reaction was terminated by cooling on ice and the sample was applied to a column (0.25 x 6 cm) of Sephadex G-75 (fine) and eluted with a buffer solution containing 100 mM Tris-HCl, pH 7.8, 10 mM MgCl,, and 4 mM 2-mercaptoethanol.
Fractions were collected in scintillat,ion vials, and radioactivit'y was measured after addition of 15 ml of Bray's solution (21).
[y-32P]GTP Incorporation-The reaction mixtures (0.2 ml) contained 100 m&r Tris-HCl buffer, pH 7. 8  PPi. 130vine serum albumin (0.2 mg) was added, and the precipitate collected by centrifugation at 12,000 X g for 20 min. The pellet was dissolved in 0.2 ml of cold 0.1 M NaOH and the RNA again precipitated with the trichloroacetic acid-PPi solution. The precipitate was collected on a Whatman GF/C glass filter, washed with approximately 20 ml of the trichloroacetic acid-Pl'i solution, and dried. The radioactivity was measured by liquid scintillation spectrometry.
DEgE-cellulose Clzromatography-The heated fraction was dialyzed against 12 liters of a standard buffer solution containing 50 rnM Tris-HCl, pH 7.5, 5 rn>l MgC12, and 5 mM 2-mercaptoethanol for 3 to 4 hours until the conductivity of a 5 x lo* dilution was less than 0.1 part, per million as NaCl.
The sample was then applied at a rate of 3 ml per min to a column (4 x 20 cm) of DEAE-cellulose previously equilibrated with standard buffer. The column was washed successively with 50 ml of standard buffer, 500 ml of the standard buffer solution containing 0.05 1\~ NaCl, 200 ml of the standard buffer solution containing 0.1 ELI NaCl, and 200 ml of the st,andard buffer solution containing 0.2 M NaCl.
A flow rate of 3 ml per min was maintained by slight positive pressure. HFI was eluted with the standard buffer solution containing 0.2 af NaCl and the fractions containing factor activity were pooled (I1EAE-cellulose fraction, 175 ml).

RESULTS
QAE-Sephadez Chromatograplzy-The DEAE-cellulose fraction was dialyzed for 2 hours against 4 liters of the standard buffer bolution containing 70 ml1 (NI-IJnSO4 and applied to a column (2 x 20 cm) of &Al!:-Sephadcx h-50 previously equilibrated with the same buffer solution.
A linear gradient of 100 mIw to 300 ma< (NHJ,SO, in a total volume of 450 ml of the standard buffer solution was then applied, and a flow rate of 15 ml per hour was maintained by slight positive pressure.
HFI activity was found in a single peak eluting between 170 and 225 mM (NHJ2S04; the bulk of the contaminating proteins eluted at the beginning of the salt gradient. In order to concentrate the factor the active fractions (120 ml) were pooled, dialyzed against 2 liters of the standard buffer solution containing 70 mM (NH4)&04, and applied at 10 ml per hour to a column (1 X 2 cm) of QAE-Sephadex 4-50. I-IF1 was eluted from the column with 7 to 10 ml of 1 M (NHJ&Oh in standard buffer and concentrated by dialysis against Sephadex G-200 (QAE-Sephadex fraction, 1.5 ml). PuriJication oj Host Factor I Cells-HF1 is found in approximately equal quantities in phage Zo?ae Sedimentation-The &BE-Sephadex fraction was dialyzed Qp infected or uninfected E. coli (I).
Uninfect,ed E. coli Q13 was against 0.2 M (NH&S04 in the standard buffer solution for 2 the source of HF1 for the purification procedure described here.
hours. Aliquots of 0.2 to 0.4 ml were layered on linear gradients The bacteria were grown to a concentration of 1 x lo9 cells per ml in 50 liters of a medium containing (per liter) 1.21 g of Tris base, 5.0 g of NaCl, 1.0 g of NH&l, 52 mg of NaJIP04.7Hz0, 100 mg of MgS04, 10 g of casamino acids, 10 ml of glycerol, and 2.5 ml of 1.0 w HCl.
The cells were harvested in a Sharples continous flow centrifuge, frozen without washing, and stored at -20". Unless otherwise indicated, subsequent operations were carried out at O-4".
Preparation of Crude E&act-Frozen cells (100 g) and cold alumina (200 g) were ground in a chilled mortar.
The cell paste was suspended by gradual addition of 400 ml of a buffer solution containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCL, 5 mM 2-mercaptoethanol, and 1 M NaCl.
The suspension was then centrifuged at 30,000 X g for 5 min. The supernatant was retained (crude fraction, 360 ml).
Liquid Polymer Fractionation-To 360 ml of the crude extract were added with mixing 42 ml of 20% (w/w) dextran 500, 112 ml of 30% (w/w) polyethylene glycol, and 61 g of NaCl. Mixing was continued for 1 hour, and the suspension was then centrifuged at 30,000 X g for 5 min. The upper phase was retained (phase fraction, 415 ml).
Heat Treatment-The phase fraction was heated in a boiling water bath with mixing until the temperature of the suspension reached 82-85" (5 to '7 min) and then immediately cooled on ice. The suspension was centrifuged at 30,000 x g for 15 min, and the supernatant was retained (heated fraction, 355 ml).
Issue of February 10, 1972 Frame de Fernandez, Hayward, and August Electrophoresis was carried out as described under "Methods." The direction of migration was from top to bottom. of 5 to 20% glycerol in standard buffer solution containing 0.2 M (NH&SO* and centrifuged in an SW 65 rotor at 50,000 rpm for 17 to 20 hours. Fractions (0.25 ml) were collected from the bottom of the tube. Almost all of the protein sedimented as a single component coincident with factor activity (Fig. 1). The active fractions were pooled (glycerol gradient fraction, 3.5 ml).

Comments on Pkjication
Procedure-A summary of the purification procedure is shown in Table I. Precise determination of factor activity in the crude and phase fractions was not possible because of inhibitors of the reaction present in these fractions. The purified HFr (glycerol gradient fraction) was stable for at least 1 month at O-4". However, factor activity is rapidly lost at low concentration (less than 10 pg per ml of protein). Because HFr is heat stable, it is possible to remove contaminating proteins by heat treatment at an early step in the purification procedure without significant loss of activity. However, to determine whether heat treatment modified the physical properties of the factor, a second purification procedure, avoiding the heat step, has been developed.
This procedure, utilizing phosphocellulose chromatography, is reported elsewhere (22). The specific activity of the purified factor, its molecular weight, elution from QAE-Sephadex, and migration during SDS-polyacrylamide gel electrophoresis were the same for both preparations.
Concentration of HFr by (NH&S04 precipitation was avoided since this invariably resulted in a loss of activity.

Properties of Host Factor I
Protein Purity-Factor activity was found coincident with protein in the fractions recovered from zonal centrifugation, the final step in the purification procedure (Fig. 1). Upon analysis of this purified fraction by SDS-polyacrylamide gel electrophoresis only a single protein band was detected (Fig. 2). This protein was the major component of the QAE-Sephadex fraction as well. The purified factor was free of RNase activity as measured by sedi- The results are the average of four determinations using three different factor preparations.
Molar ratios were calculated relative to alanine.
The number of residues per chain was calculated assuming a molecular weight of 12,500; the values have been rounded to the nearest integer.
Tryptophan and cysteine were not determined. Molecular ll'eight-The apparent molecular weight of the active factor is 70,000 to 80,000 as measured by zone sedimentation in staudard buffer containing 0.2 M (NHJ2S04 (Fig. 1). A similar value was obtained by means of gel filtration (22). The molecular weight of the single polypeptide resolved by SDS-polyacrylamide gel clectrophoresis was 12,000 to 13,000, as determined by the method of Shapiro et al. (20). This suggests that the active factor conta,ins a subunit structure of six polypeptide chains.
Chemical Compositio7z-The heat resistance of the factor led us to consider the possibility that it might have a chemical composition different from that of a simple protein.
However, no unusual constituents were detected by amino acid analysis (Table  II).
The absence of carbohydrates was confirmed by gas-liquid chromatogra,phy, indicating that nucleotides were not present in the pure HIpI preparation.
Under the experimental conditions, 1 n-role of ribose or deoxyribose per mole of factor (mol wt 12,500) could have been detected.
The density of the factor was 1. that of a protein, as determined by density gradient sedimentation in CsCI.
Studies on Role of Host Factor I Requirement for Host Factor I-Replication of &fi RNA is completely dependent on HF1 (Fig. 3). The small amount of RNA synthesized after prolonged incubation in the absence of factor is 6 S RNA, which can be replicated in the absence of host factors (23). Synthesis of this RNA is presumably due to contamination of the enzyme preparation with a small amount of 6 S RNA, since it is also observed when the Qp RNA template is omitted from the reaction mixture.
At low concentrations of HFI, the rate of synthesis is proportional to factor concentration.
The kinetics of the reaction, how ever, are complex, with an initial lag and an early termination of the reaction.
Since the initial lag is reduced by increased HFI, it is possible that the factor is involved in an early rate-limiting step.
Requirement for Host Factor I as Function of RNA Concentration-The concentration of factor required for the maximum rate of synthesis varies according to the amount of RNA added to the reaction, but is independent of enzyme concentration (Fig. 4). The amount of factor required for half-maximal activity was the same over a 5-fold range of enzyme concentrations when the amount of RN-4 in the reaction was held constant (Fig. 4A). With different concentrations of RN.4 and constant enzyme, the amount of factor required for half-maximal activity was proportional to the amount of RNA (Fig. 4B). These data suggest that the role of HF1 in the reaction is related to Rn'h, not to enzyme. Assuming that all added RN-4 is active with respect to factor it can be estimated from extrapolation of these curves that 1 molecule of HF1 (mol wt 75,000) is required per molecule of Qp RNA.
Binding of Host Factor I to Single stranded RNA-The association of HF1 and RS.1 was analyzed by zone sedimentation. Factor alone sediments at approximately 5 S, but when mixed with Q/3 RNA the active factor cosediments with the RNA (Fig.  5 6. Analysis of the association of I-IF1 with RiVA. The experiments were performed as described in Fig. 5 except that samples containing R23 and f2 RNA were centrifuged at 60,000 rpm for 2+ hours and those containing rRNA and reovirus RNA were centrifuged at 50,000 rpm for 4 hours. O---O, absorbance; A---A, HFr act.ivity. This binding is not limited to template RNA since HF1 also binds to F. coli rRNA and phage R23 and f2 RNA (Fig. 6). However, the reaction does appear to be specific for single stranded RSA.
No binding was observed with double stranded reovirua RNA except for a faster sedimenting minor component which behaved as single stranded RNA.
These experiments with nontemplate R.NA suggest that factor binding is readily reversible, since factor activity could be measured in the Q/3 RNXdirected reaction.
Approximately 75% of t,he factor activity was recovered.
Binding was not detected with DNA, whether single stranded bacteriophage fd DNA, or double stranded PBSX or X DNA (Fig. 7). E$ect of Incubation of Q,B RNA with Host Factor I-The possibility that HF, causes an irreversible change in the template RNA was tested by incubating Qp RNA with the factor alone, with HF, and HFI1, or with HF1, HFI1, and the Qfi RNA polymerase. The RNA was then treated by phenol extraction to remove the added protein and tested for template activity in the presence and absence of HF1 (Table III).
In every case HF, was required for template activity, despite the previous treatment. This was true even when the RNA was first incubated with both factors, substrates (GTP, CTP, UTP), and polymerase, conditions which allow synthesis of a poly(G) sequence.4 This suggests that HF1 activity does not involve an irreversible alteration of RNA, such as a change in primary structure.
Effect of Host Factor I on Initiation of Synthesis--Successive steps in the synthesis of Q/3 RNA are the association of enzyme and Qfl RNA, the initiation of synthesis, and chain elongation. As the nonspecific binding of RNA by the enzyme at 0" and in t,he absence of substrates does not require the presence of HF1 (3,24), it can be speculated that factor is required for a subsequent step leading to the initiation of synthesis or to chain elangation.
An analysis of the requirement for HF, in the initiation of synthesis has been performed by studies of pyrophosphate exchange, synthesis of a 5'.terminal polynucleotide, and [-y-V-GTP incorporat'ion.
The pyrophosphate exchange reaction is completely dependent on HFI (Fig. 8) suggesting that the factor is required for chain initiation.
Since it has not been proven, however, that the pyro-4 W'. S. Hayward and P. Trown, unpublished observations.  phosphate exchange reaction can discriminate bet)ween chain initiation and elongation, other more direct assays were carried out. Synthesis of a limited sequence at the 5' terminus of the complementary strand can be analyzed by adding only AT!' and GTP as substrates for the reaction since the first 14 nucleotides contain only guanylale and adenylate residues (25). After incubation in the presence or absence of HF,, the reaction mixtures TT-ere cooled on ice and passed through Sephadex columns to separate the enzyme-template-nascent RNA complexes from the labeled substrate.
Approximately 3.5 pmoles of [3H]GRIP Il-vere incorporated into the oligonucleotide product in the presence of HF1, while less than 0.2 pmole was incorporated when the factor was omitted (Fig. 9) This suggests that neither phosphodiester bond formation nor irreversible binding of 5'-terminal GTP occurs in the absence of factor I.

DISCUSSION
Several lines of evidence suggest that the role of HF1 in the Qfi RN9 polymerase reaction is related specifically to the use of Qp RNA as template.
As previously reported, factors are not required with any other template for the enzyme (3). Moreover, the factor appears to act directly on the RNA, rather than functioning as an enzyme subunit, since the concentration of I-IF1 required in the reaction is stoichiometric with the concentration of RNA, but is independent of enzyme concentration.
The factor binds t'o RNA even in the absence of enzyme or other components of the reaction, and this binding appears to be specific for single stranded RNA since it did not occur with double stranded reovirus RNA or with DNA.
This observation suggests that HF1 acts as a positive control element regulating the utilization of template in the reaction.
This may be related to the synthesis in the reaction predominantly of Q/I RNA, not the complementary strand (3,26,27). At this time the only known difference in the utilization of Qfl RNA or its complement as template is the requirement for factors with Qp RNA but not with the complement (3). HFI appears to act at an early step in the Qfl polymerase reaction. The factor is absolutely required for pyrophosphate exchange, chain initiation (incorporation of [y-32P]GTP), and polymerization when Qfl RNA is template. Thus it seems likely that the factor acts at some step prior to the initiation of synthesis. The apparent first step in the polymerase reaction is the binding of RNA by the enzyme.
This reaction takes place in the absence of factors, but occurs at multiple sites on nontemplate as well as template RNA and is readily reversible.
It has recently been found, however, that if the enzyme-RNA complex is incubated with the two host factors and GTP, an irreversible complex is formed at a single site on template RNA (28). It is thus speculated that the factors are involved in the formation of a specific initiation complex of enzyme, RNA, factors, and GTP. Q/3 RNA is known to contain extensive regions of hydrogenbonded duplex structures (29). One possible model for IIFI action is that the factor mediates a change in the secondary or ter-Gary structure of the RN,4 which allows the enzyme to bind to a site that is unavailable in the folded RNA molecule.
Since only 1 molecule of HFI per molecule of RNA is required for synthesis, such an interaction would appear to involve only limited regions of the RNA, presumably at or near the enzyme-binding site.