RESUMEN
Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Escherichia coli , Duplicado del Terminal Largo de VIH , VIH-1/genética , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Endorribonucleasas/genética , Escherichia coli/enzimología , Productos del Gen tat/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , ARN Viral/genética , Ribonucleasa III , Homología de Secuencia de Ácido Nucleico , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.
Asunto(s)
VIH-1/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Análisis Mutacional de ADN , Genes , Células HeLa , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , ARN Viral/ultraestructura , Alineación de Secuencia , Eliminación de Secuencia , Linfocitos T/metabolismo , Linfocitos T/microbiologíaRESUMEN
TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.
Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/química , Imitación Molecular , ARN Viral/química , Proteínas de Unión al ARN/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Péptidos/química , Conformación Proteica , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.
Asunto(s)
Bleomicina/farmacología , Farmacorresistencia Microbiana/genética , Fleomicinas/farmacología , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Fúngicos , Clonación Molecular/métodos , ADN de Hongos/genética , Genes Fúngicos , Vectores Genéticos , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
The protein coded by a bleomycin-resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E. coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin-binding protein.
Asunto(s)
Bleomicina/metabolismo , Proteínas Portadoras/farmacología , ADN/metabolismo , Actinomycetales/genética , Bleomicina/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , ADN Recombinante , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Fluorescencia , Concentración OsmolarRESUMEN
TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR. Copyright 1995 S. Karger AG, Basel
RESUMEN
The regulation of HIV expression is controlled by the activity of the Long Terminal Repeat (LTR). Trans-activation by the virally encoded Tat protein is one of the main mechanisms of LTR activation. Tat binds to its target, TAR RNA, and cellular proteins that bind the LTR, Tat, or TAR RNA are important components of the trans-activation process. We will review the factors that have been characterized for a possible involvement in this mechanism. Whereas LTR binding proteins consist of Sp1 and TBP, a large number of factors that bind TAR RNA have been isolated. We have previously cloned two of them by RNA probe recognition: TRBP and La. We have shown that the in vitro and in vivo binding of TRBP to TAR RNA correlates with a constant expression of the protein during HIV-1 infection. Several proteins that interact with Tat have mainly positive, but some negative, effects on trans-activation. Genetic studies have defined that human chromosome 12 encodes a protein that will allow trans-activation in rodent cells. The binding and the functional data about these proteins suggest sequential steps for the Tat trans-activation mechanism. Each of these intracellular molecular events could be the target for molecular intervention against the virus.
Asunto(s)
Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/genética , VIH-1/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Viral/genética , ADN Viral/metabolismo , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Unión Proteica , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Oncogene MYC is highly expressed in many human cancers and functions as a global regulator of ribosome biogenesis. Previously, we reported that ribosomal protein (RP) L11 binds to c-Myc and inhibits its transcriptional activity in response to ribosomal stress. Here, we show that RPL5, co-operatively with RPL11, guides the RNA-induced silencing complex (RISC) to c-Myc mRNA and mediates the degradation of the mRNA, consequently leading to inhibition of c-Myc activity. Knocking down of RPL5 induced c-Myc expression at both mRNA and protein levels, whereas overexpression of RPL5 suppressed c-Myc expression and activity. Immunoprecipitation revealed that RPL5 binds to 3'UTR of c-Myc mRNA and two subunits of RISC, TRBP (HIV-1 TAR RNA-binding protein) and Ago2, mediating the targeting of c-Myc mRNA by miRNAs. Interestingly, RPL5 and RPL11 co-resided on c-Myc mRNA and suppressed c-Myc expression co-operatively. These findings uncover a mechanism by which these two RPs can co-operatively suppress c-Myc expression, allowing a tightly controlled ribosome biogenesis in cells.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Regiones no Traducidas 3' , Proteínas Argonautas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Medios de Cultivo/farmacología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismoAsunto(s)
Lentivirus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Regulación Viral de la Expresión Génica , VIH/genética , VIH/fisiología , Lentivirus/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Viral/química , ARN Viral/genética , ARN Viral/metabolismo , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Transcripción GenéticaAsunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Unión al ADN/genética , Frecuencia de los Genes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Mutación/genética , Factores de Transcripción/genética , Análisis Mutacional de ADN , Proteína 2 de la Respuesta de Crecimiento Precoz , Femenino , Pruebas Genéticas , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/clasificación , Humanos , Masculino , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Dedos de ZincAsunto(s)
Productos del Gen tat/fisiología , VIH-1/genética , Proteínas de Saccharomyces cerevisiae , Transactivadores/fisiología , Acetiltransferasas/metabolismo , Síndrome de Inmunodeficiencia Adquirida/terapia , Ciclina T , Ciclinas/fisiología , Productos del Gen tat/antagonistas & inhibidores , VIH-1/fisiología , Histona Acetiltransferasas , Humanos , Proteínas de Unión al ARN/fisiología , Activación Transcripcional , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.
Asunto(s)
Bleomicina/farmacología , Elementos Transponibles de ADN , Genes Dominantes , Genes Fúngicos , Fleomicinas/farmacología , Saccharomyces cerevisiae/genética , Clonación Molecular , Resistencia a Medicamentos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that activates transcription from the long terminal repeats. The target for transactivation is termed the trans-acting responsive (TAR) element. TAR has an extensively folded RNA secondary structure and is present at the 5' end of all viral mRNAs. Considerable similarities exist between both Tat and TAR of the two viruses. The TAR element of HIV-2 (TAR-2) resembles a tandem duplication of the TAR-1 hairpin structure. Tat-2 conserves many of the protein domains in Tat-1, although it is slightly larger than its counterpart. Given the similarity between the two Tat proteins, it is somewhat unexpected that HIV-2 Tat (Tat-2) only poorly activates the heterologous TAR-1 element. Here, we tested whether the duplicated structure of TAR-2 is required for full Tat-2 activity. We show that the addition of a second TAR hairpin to TAR-1 increased its Tat-2 responsiveness by 3-fold.
Asunto(s)
Productos del Gen tat/metabolismo , VIH-2/genética , ARN Viral/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transactivadores/metabolismo , Activación Transcripcional , Secuencia de Bases , Exones , VIH-1/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos , Plásmidos , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.
Asunto(s)
Bleomicina/farmacología , Resistencia a Medicamentos/genética , Genes Dominantes , Marcadores Genéticos , Fleomicinas/farmacología , Animales , Línea Celular , Cricetinae , Cricetulus , ADN/genética , Hibridación de Ácido Nucleico , Plásmidos , TransfecciónRESUMEN
Human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat activates the expression of its viral long terminal repeat (LTR) through a target transactivation-responsive element termed TAR. We have constructed cell lines that constitutively express the HIV-1 Tat protein. Analyses of nuclear proteins from these cells and from matched control cells that do not express Tat have identified three proteins that bind to a radiolabeled HIV-1 TAR RNA probe. These polypeptides are 100 kDa, 62 kDa, and 46 kDa in size. Competition experiments using a wild-type TAR RNA sequence, a biologically inactive mutant sequence of TAR, and an unrelated RNA species demonstrated that these proteins show higher binding affinity to wild-type TAR than to the other two non-trans-activatable sequences. We hypothesize that these cellular proteins may mediate a function necessary in Tat-dependent activation of the LTR. The fact that no differences were seen in the binding profiles of nuclear proteins to TAR RNA in Tat-producing and Tat-nonproducing cells suggests that Tat does not directly interact with TAR.
Asunto(s)
Genes Virales , Genes tat , VIH-1/genética , ARN Viral/genética , Activación Transcripcional , Secuencia de Bases , Proteínas Portadoras/aislamiento & purificación , Células HeLa/metabolismo , Humanos , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Sondas ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Proteínas de Unión al ARN , Transcripción Genética , TransfecciónRESUMEN
Cellular context is an important determinant for the activity of Tat, the trans-activator of human immunodeficiency virus (HIV). We have investigated HIV-1 promoter expression and trans-activation in Saccharomyces cerevisiae to provide clues about the limiting steps for Tat activity in this organism. A minimal 43-nucleotide HIV promoter (HIV43) has the activity of a weak yeast promoter in the presence or absence of various enhancer binding sites (bs), whereas the entire long terminal repeat is not expressed. None of these constructs could be trans-activated by Tat. Fusion proteins Gal4 binding domain (BD)-Tat48 and Gal4BD-Tat72 are active with different efficiencies on various yeast promoters that have Gal4 bs. They have 70 and 50% of Gal4 wild type activity on hybrid HIV promoters fused to Gal4 bs only in the presence of AP1 bs. This study shows that trans-activation of the HIV-1 promoter by Tat occurs in yeast when Tat is targeted to the promoter and a functional enhancer activity is present. Sp1 function and Tat transfer from the RNA to the promoter are two major elements for in vivo trans-activation of HIV-1 that are defective in S. cerevisiae but can be replaced by functional equivalents.
Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Transcripción Genética , Activación Transcripcional , Secuencia de Bases , Replicación del ADN , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Regulación Viral de la Expresión Génica , Duplicado del Terminal Largo de VIH/genética , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fleomicinas/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/virología , Factor de Transcripción Sp1/metabolismo , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Integración Viral , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Replication of HIV-1 requires Tat, which stimulates gene expression through a target sequence, TAR. It is known that TAR is a Tat-responsive target. Since Tat increases transcriptional initiations from the HIV-1 LTR promoter, it is unclear mechanistically how Tat utilizes an RNA target. Here we show that TAR RNA is only one component of the Tat-responsive target. Efficient Tat trans-activation was observed only when TAR was present in conjunction with the HIV-1 LTR NF-kappa B/SP1 DNA sequences. TAR RNA outside of this context produced a suboptimal Tat response. We propose that TAR RNA serves an attachment function directing Tat to the LTR. A Tat protein engineered to interact with LTR DNA could trans-activate through a TAR-independent mechanism. This suggests that Tat also has a DNA target.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen tat/fisiología , VIH-1/genética , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Transactivadores/fisiología , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , ADN Viral/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , ARN Viral/genética , Proteínas Recombinantes de Fusión , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
Translational effects of the RNA leader and Tat protein of human immunodeficiency virus type 1 (HIV-1) were investigated in rabbit reticulocyte lysate. Hybrid RNA species with natural or mutated HIV-1 leader fused to human interferon- gamma mRNA were produced in vitro from recombinant plasmids. HIV-1 leader RNA was found to inhibit translation through two mechanisms. A 3-fold trans-inhibition of translation was demonstrated by mixing hybrid HIV-1 leader RNA with indicator interferon mRNA. By comparison, HIV-1 leader caused a 50-fold cis-inhibition in lysate in which two trans-inhibitory factors, double-stranded RNA-dependent protein kinase and (2'-5')oligoadenylate synthetase, were suppressed. In contrast, purified HIV-1 Tat protein produced in Escherichia coli enhanced by 4-fold translation from HIV-1 leader-interferon mRNA but not from interferon mRNA lacking HIV sequences or from total poly(A)+ RNA. Translation of mRNA containing either a single base substitution in the loop of the "trans-acting responsive" sequence (TAR) or an alternative stem-loop in TAR was nevertheless stimulated by Tat. The enhancement of translation by Tat was largely due to relief of cis-inhibition, since the effect was found even in lysate in which double-stranded RNA-dependent protein kinase was inhibited with 2-aminopurine. These results suggest that translation is an important level of control in the replication cycle of HIV-1.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen tat/genética , VIH-1/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Productos del Gen tat/biosíntesis , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Cinética , Mutación , Plásmidos , Conejos , Proteínas Recombinantes de Fusión/biosíntesis , Reticulocitos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
A technique to detect RNA-binding proteins (RBP) involving hybridization of RNA probe to proteins transferred to a membrane was used to study RBP in different mammalian cells and in cells after genotoxic stress. With this approach, up to 13 proteins of different sizes were detected in crude nuclear extracts by using a viral RNA probe consisting of the trans-activation-responsive (TAR) element of human immunodeficiency virus type 1 (HIV-1). The TAR RNA probe contains a stem-loop structure found in nascent HIV-1 transcripts. A G+C-rich probe with similar structure also bound to many of these RBP. Only a 102-kDa protein strongly bound to other RNA probes lacking this structure, while a probe with an A+U-rich stem-loop structure fail to bind most RBP, thus indicating a RNA secondary structure preference. The expression of these RBP varied substantially in nine different human and hamster cell lines, with no detectable RBP in two human myeloid lines. Evidence for induction of these RBP was found in six of seven lines after treatment with DNA-damaging agents; UV radiation was the most effective agent. In Chinese hamster ovary cells, which showed the strongest response, all five RBP present in untreated cells rapidly increased in activity after UV irradiation, and eight additional RBP were detected. The induction of these RBP by DNA-damaging agents indicates one or more possible roles for these proteins in the cellular response to genotoxic stress and in viral activation after such stress.
Asunto(s)
Daño del ADN/fisiología , Regulación de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Animales , Células CHO/efectos de la radiación , Línea Celular , Cricetinae , Rayos gamma , Humanos , Metilmetanosulfonato/farmacología , Proteínas Nucleares/metabolismo , Unión Proteica , ARN/metabolismo , Especificidad de la Especie , Rayos Ultravioleta , Xerodermia Pigmentosa/metabolismoRESUMEN
We have characterized the in vivo and in vitro binding of human La protein to the human immunodeficiency virus type 1 (HIV-1) leader RNA, the trans-activation response element (TAR). In immunoprecipitation studies using anti-La serum, La-TAR ribonucleoproteins were recovered from HIV-1-infected lymphocytes. Further characterization of this interaction revealed that La has preference for the TAR stem. However, TAR RNA recognition tolerated changes in the primary sequence of the stem as long as the secondary structure was conserved. This structural aspect of La-TAR recognition was confirmed in competition studies in which certain homopolymers influenced complex formation while other single-stranded and double-stranded RNAs had no effect. Deletion mutants of recombinant La protein were used to demonstrate that the residues responsible for binding to polymerase III precursor transcripts overlapped the binding domain for the TAR leader RNA. This finding of a direct interaction between La and TAR has functional implications for translational regulation of HIV-1 mRNAs as demonstrated in the accompanying report (Y. V. Svitkin, A. Pause, and N. Sonenberg, J. Virol. 68:7001-7007, 1994).