Biomedical subjects

M Laughrea

Publications and source records attributed to M Laughrea.

At least 19 recordsLinked to original sources

PubMed2001

Role of distal zinc finger of nucleocapsid protein in genomic RNA dimerization of human immunodeficiency virus type 1; no role for the palindrome crowning the R-U5 hairpin.

Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein (NC) was characterized by nondenaturing gel electrophoresis. Mutations in the C-terminal, the N-terminal, and the linker regions had no effect on genomic RNA dimerization [they are R7R10K11S, P31L, R32G, S3(32-34), and K59L], while a C36S/C39S mutation in the distal zinc knuckle (Cys-His box or zinc finger) inhibited genome dimerization as much as disrupting the kissing-loop domain. The four mutations which inhibited tRNA(Lys3) genomic placement (i.e., the in vivo placement of tRNA(Lys3) on the primer binding site) had no effect on genome dimerization. Among five mutations which inhibited genome packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA(Lys3) placement (two processes which do not require mature NC) but have little influence on genome dimerization and 2-base extension of tRNA(Lys3) (two processes which are likely to require mature NC). It has been suggested, based on electron microscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulates genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpin or kissing-loop domain; in another mutant, we duplicated AGCU81. The loss of AGCU81 reduced dimerization by 2.5 +/- 4%; its duplication increased it by 3 +/- 6%. Dissociation temperature was left unchanged. We reach two conclusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differentially influenced by NC sequence: it is Zn finger dependent but independent of the basic nature of the N-terminal and linker subdomains. We propose that the NC regions implicated in 2-base extension of tRNA(Lys3) are required for a second (maturation) step of tRNA placement. Genome dimerization and mature tRNA placement would then become two RNA-RNA interactions sharing similar NC sequence requirements.

Amino Acid Sequence↗

PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗

PubMed2000

Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging.

The kissing-loop domain (KLD) encompasses a stem-loop, named kissing-loop or dimerization initiation site (DIS) hairpin (nucleotides [nt] 248 to 270 in the human immunodeficiency virus type 1 strains HIV-1(Lai) and HIV-1(Hxb2)), seated on top of a 12-nt stem-internal loop called stem-loop B (nt 243 to 247 and 271 to 277). Destroying stem-loop B reduced genome dimerization by approximately 50% and proviral DNA synthesis by approximately 85% and left unchanged the dissociation temperature of dimeric genomic RNA. The most affected step of reverse transcription was plus-strand DNA transfer, which was reduced by approximately 80%. Deleting nt 241 to 256 or 200 to 256 did not reduce genome dimerization significantly more than the destruction of stem-loop B or the DIS hairpin. We conclude that the KLD is nonmodular: mutations in stem-loop B and in the DIS hairpin have similar effects on genome dimerization, reverse transcription, and encapsidation and are also "nonadditive"; i.e., a larger deletion spanning both of these structures has the same effects on genome dimerization and encapsidation as if stem-loop B strongly impacted DIS hairpin function and vice versa. A C258G transversion in the palindrome of the kissing-loop reduced genome dimerization by approximately 50% and viral infectivity by approximately 1.4 log. Two mutations, CGCG261-->UUAA261 (creating a weaker palindrome) and a Delta241-256 suppressor mutation, were each able to reduce genome dimerization but leave genome packaging unaffected.

Animals↗

PubMed1999

Variant effects of non-native kissing-loop hairpin palindromes on HIV replication and HIV RNA dimerization: role of stem-loop B in HIV replication and HIV RNA dimerization.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. In vitro synthesized RNAs from human immunodeficiency virus type 1 (HIV-1) can form loose or tight dimers depending on whether their respective kissing-loop hairpins (nts 248-270 in HIV-1Lai) bond via their hexameric autocomplementary sequences (ACS), also called palindromes, or via the ACS and stem sequences [Laughrea, M., and Jetté, L. (1996) Biochemistry 35, 1589-1598]. To understand the role of the ACS in HIV-1 replication and in the formation and stability of HIV-1 RNA dimers, we replaced the central CGCG261(or tetramer) of the HIV-1Lai ACS by two other HIV-1 tetramers (UGCA/UGCG), four non-HIV-1 tetramers [GUAC, UUAA (respectively found in HIV-2Rod and SIVmnd), GGCC and AGCU (absent from HIV and SIV viruses)], or GGCG, a nonpalindromic tetramer. The infectivity of GGCC, GUAC, and UGCA viruses was unchanged or insignificantly decreased; the infectivity of AGCU and UGCG viruses was decreased by 80%; the infectivity of UUAA and GGCG viruses was decreased by 92-98%. Thus, the four non-HIV-1 palindromes yielded phenotypes ranging from wild-type to as defective as a virus bearing a nonpalindrome. Studies of in vitro synthesized HIV-1 RNAs were generally consistent with in vivo results, specifically: (i) loose dimerization of GGCC and GUAC RNAs, but not of UUAA and AGCU RNAs, was influenced by the 3' DLS (a sequence located downstream of the 5' splice junction) in a way expected for a wild-type ACS; (ii) the 3' DLS strongly reduced tight dimerization of UUAA and AGCU RNAs, but not of GGCC and GUAC RNAs. We conclude that HIV-1 is sensitive to the ACS sequence without discriminating against all nonnative ACS: GGCC/GUAC, but not AGCU/UUAA, are good substitutes for the prevalent CGCG/UGCA native tetramers and better substitutes than the very rare UGCG native tetramer. The correlation between in vivo and in vitro results suggests that in vitro assays measure parameters of in vivo relevance. Deletion of CUCGG247 (the 5' strand of stem-loop B) decreased the replicative capacity by more than 99.9% and metamorphosed the 3' DLS into an inhibitor of the loose dimerization of HIV-1 RNA.

Base Composition↗

PubMed1999

Deletion mutagenesis within the dimerization initiation site of human immunodeficiency virus type 1 results in delayed processing of the p2 peptide from precursor proteins.

Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins.

Animals↗

PubMed1999

Mutations within four distinct gag proteins are required to restore replication of human immunodeficiency virus type 1 after deletion mutagenesis within the dimerization initiation site.

Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only marginally impaired encapsidation while the BH10-LD3 deletion caused a severe deficit in this regard.

Amino Acid Sequence↗

PubMed1998

Compensatory point mutations in the human immunodeficiency virus type 1 Gag region that are distal from deletion mutations in the dimerization initiation site can restore viral replication.

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3' end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.

Amino Acid Sequence↗

PubMed1997

Sequence elements downstream of the human immunodeficiency virus type 1 long terminal repeat are required for efficient viral gene transcription.

We investigated the role of a 54-nucleotide region (+200 to +253) located downstream of the HIV-1 long terminal repeat (LTR) on virus gene expression and found, using RT-PCR and p24 CA analysis, that deletion of this region inhibited synthesis of both viral RNA and protein. CAT assays showed that these results were attributable to decreased transcription efficiency of the HIV-1 LTR and not to the stability of the RNA transcripts produced. Further deletional analysis and transfection studies showed that the most important sequences with regard to proviral DNA expression were located between nucleotide positions +218 and +237. Furthermore, substitutional mutational analysis showed that a CTCTCTC sequence at positions +227 to +233, homologous to the pyrimidine-rich initiator (Inr) region found in several promoters, was required for efficient production of both viral RNA and protein. Deletion of the sequence +200 to +217, homologous to the interferon-stimulated response element (ISRE), resulted in impaired LTR promoter activity and decreased synthesis of viral RNA and protein. However, when the latter region was replaced by homologous ISRE sequences from an interferon-stimulated gene (ISG-54), an even more severe effect on HIV gene expression and replication was observed, suggesting that ISRE-like sequences in HIV act differently from homologous sequences in interferon-responsive cellular genes.

Animals↗

PubMed1997

HIV-1 genome dimerization: kissing-loop hairpin dictates whether nucleotides downstream of the 5' splice junction contribute to loose and tight dimerization of human immunodeficiency virus RNA.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Adjacent genomic RNAs from human immunodeficiency virus type 1 (HIV-1) can form loose or tight dimers depending on whether their respective kissing-loop hairpins (nts 248-270 in HIV-1Lai) bond via their autocomplementary sequences (ACS) or via the ACS and stem sequences [Laughrea, M., & Jetté, L. (1996a) Biochemistry 35, 1589-1598]. Loose dimers from HIV-1Mal, but not HIV-1Lai, are stabilized by a sequence (3'DLS) located downstream of the 5' splice junction [Laughrea, M., & Jetté, L. (1996b) Biochemistry 35; 9366-9374]. To understand the ACS-3'DLS interplay in the formation and stability of loose and tight HIV-1 RNA dimers, we replaced the ACS of HIV-1Lai (GCGCGC262) by GUGCAC, GUGCGC (two alternative HIV-1 ACS), or GAGCUC (a non-HIV ACS). For each mutant, RNAs truncated immediately upstream or downstream of the 3'DLS were prepared; their ability to dimerize and their thermal stabilities were compared. The results suggest that the ACS determines whether the 3'DLS participates in RNA dimerization: (1) GAGCUC262 led to poorly stable loose dimers due to the inability of the 3'DLS to stabilize them (the 3'DLS stabilized the GUGCAC and GUGCGC RNAs); (2) GAGCUC262 led to poor formation of tight dimers, due to an inhibitory effect of the 3'DLS (the 3'DLS had little effect on the tight dimerization of the GUGCAC, GUGCGC and GCGCGC RNAs). The results indicate that communication exists between HIV-1 RNA sequences respectively located upstream and downstream of the 5' splice junction; they are consistent with the idea that the 3'DLS plays two ACS-dependent roles in the dimerization process: loose dimer stabilization in HIV-1 RNAs bearing an HIV ACS (unless the ACS already conferred a thermostability equal or superior to that offered by the 3'DLS), and inhibition of tight dimer formation in an HIV-1 RNA bearing a non-HIV ACS.

Base Sequence↗

PubMed1997

Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization.

A stem-loop termed the kissing-loop hairpin is one of the most highly conserved structures within the leader of human immunodeficiency virus type 1 (HIV-1) and chimpanzee immunodeficiency virus genomic RNA. Because it plays a key role in the in vitro dimerization of short HIV-1 RNA transcripts (M. Laughrea and L. Jette, Biochemistry 35:1589-1598, 1996, and references therein; M. Laughrea and L. Jette, Biochemistry 35:9366-9374, 1996, and references therein) and because dimeric RNAs may be preferably encapsidated into the HIV-1 virus, alterations of the kissing-loop hairpin might affect the in vivo dimerization and encapsidation processes. Accordingly, substitution and deletion mutations were introduced into the kissing-loop hairpin of an infectious HIV-1 molecular clone in order to produce viruses by transfection methods. The infectivity of the resulting viruses was decreased by at least 99%, the amount of genomic RNA packaged per virus was decreased by 50 to 75%, and the proportion of dimeric genomic RNA was reduced from >80 to 40 to 50%, but the dissociation temperature of the genomic RNA was unchanged. There is evidence suggesting that the deletion mutations moderately inhibited CAp24 production but had no significant effect on RNA splicing. These results are consistent with the kissing-loop model of HIV-1 RNA dimerization. In fact, because intracellular viral RNAs are probably more concentrated in transfected cells than in cells infected by one virus and because the dimerization and encapsidation processes are concentration dependent, it is likely that much larger dimerization and encapsidation defects would have been manifested within cells infected by no more than one virus.

Animals↗

PubMed1997

Identification of sequences downstream of the primer binding site that are important for efficient replication of human immunodeficiency virus type 1.

Reverse transcription of retroviruses is initiated from an 18-nucleotide (nt) primer binding site (PBS), located within the 5' region of viral genomic RNA, to which the host cell-derived tRNA primer is annealed and also involves viral genomic sequences outside the PBS. We constructed proviral DNA clones of human immunodeficiency virus (HIV) that had selective deletions of either a 7-nt segment found immediately downstream of the PBS or an extended nontranslated 54-nt stretch located immediately downstream of the PBS and containing the aforementioned 7-nt segment. Synthesis of minus-strand strong-stop DNA was assessed with MT-4 cells infected with viruses derived from COS-7 cells that had been transfected with these various constructs. We found that similar levels of minus-strand strong-stop DNA as well as DNA produced after template switching were expressed in MT-4 cells infected with COS-7-derived wild-type viruses or with viruses that had the 7-nt segment deleted. In contrast, significantly lower levels of viral DNA were detected in MT-4 cells after infection with viruses that had deletions of the 54-nt stretch. Furthermore, the molecular clone containing the 7-nt deletion was able to replicate with wild-type kinetics, while that containing the 54-nt deletion displayed a significantly diminished capacity in this regard. Further deletion analysis showed that a 16-nt segment at the 3' end of this 54-nt segment was largely responsible for these effects. We also conducted studies to determine levels of viral mRNA in COS-7 cells that had been transfected with equivalent amounts of DNA derived from either a wild-type HIV construct or our various deletion mutants. In the case of transfections performed with the 7-nt deletion mutant and wild-type HIV DNA, high levels of viral mRNA transcripts were detected, which was not the case for the 54 nt-deletion mutant. However, these various mRNAs possessed similar stabilities, as shown through studies in which transcript formation was arrested by treatment of cells with actinomycin D. Thus, the 54-nt segment of 5' nontranslated RNA, located downstream of the PBS, is involved in efficient expression of each of viral DNA, mRNA, and infectious virus.

Binding Sites↗

PubMed1996

HIV-1 genome dimerization: formation kinetics and thermal stability of dimeric HIV-1Lai RNAs are not improved by the 1-232 and 296-790 regions flanking the kissing-loop domain.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization posits that the 233-285 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is at least the core dimerization domain of HIV-1 RNA. This region is called the kissing-loop domain. In addition, it can be argued that sequences within the 296-401 region [Paillart et al. (1994) J. Biol. Chem. 269, 27486-27493] or 5' of the primer binding site [Laughrea & Jetté (1996) Biochemistry 35, 1589-1598] might play some role in the dimerization process. Accordingly, we have studied the effect of regions 1-232 and 296-790 on the dimerization kinetics and thermal stability of HIV-1Lai RNAs containing the kissing-loop domain (HIV-1Lai is a typical representative of North American and European HIV-1 viruses). Experiments conducted at high and low ionic strength indicate that these regions have no strongly positive effect on the dimerization process. Our experiments also indicate that the kissing-loop domain of HIV-1Lai has an apparent dissociation temperature 13 degrees C higher than that of the HIV-1Mal kissing-loop domain (HIV-1Mal is a Central African virus whose kissing-loop domain has a "weak" GUGCAC autocomplementary sequence). Because the 296-401 region of HIV-1Mal RNA stabilizes dimeric RNAs by < or = 12 degrees C (Paillart et al., 1994), we infer that the contributions of sequences downstream of U295 are (at best) concealed in HIV-1Lai and in most American and European HIV-1 viruses, i.e., in viruses whose kissing-loop domain is characterized by a "strong" GCGCGC autocomplementary sequence.

Base Sequence↗

PubMed1996

Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation.

The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. The kissing-loop model of HIV-1 genome dimerization [Laughrea, M., & Jetté, L. (1994) Biochemistry 33, 13464-13474; Skripkin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 4945-4949] posits that the 248-270 region of the HIV-1 genome, by forming a hairpin and initiating dimerization through a loop-loop interaction, is the full or at least the core dimerization domain of HIV-1 RNA. Here, we show by nested deletion analysis that the 3' boundary of the HIV-1 dimerization domain is immediately downstream of hairpin 248-270 and that the isolated region 248-271 dimerizes at least as readily as longer RNAs. Among various HIV-1Lai RNA transcripts containing hairpin 248-270, all form two types of dimer, as is implicit in the kissing-loop model. The high-stability dimer resists semidenaturing conditions and the low-stability dimer cannot, which is consistent with the model. At physiological temperatures, low-stability dimers are usually formed, as if dimerization without nucleocapsid proteins corresponded to loop-loop interaction without switching from intra- to interstrand hydrogen bonding. Our results show that the 3' DLS (a sequence immediately 3' from the 5' splice junction and originally thought to be the dimerization domain of the HIV-1 genome) and adjacent nucleotides are not necessary for efficient dimerization of HIV-1Lai RNA at low and high ionic strength. Upstream of hairpin 248-270 exists another "DLS-like" sequence that we name 5' DLS: like the isolated 3' DLS, the isolated 5' DLS forms an apparently nonphysiological structure that can become substantially dimeric at high ionic strength.

Base Sequence↗

PubMed1994

A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA.

The genome of all retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNAs noncovalently linked near their 5' end. Dimerization of genomic RNA is thought to modulate several steps in the retroviral life cycle, such as recombination, translation, and encapsidation. We report the results of experiments designed to identify the 5' and 3' boundaries of the dimerization domain of the HIV-1 genome: (1) An HIV-1 RNA starting at nucleotide 252 or at other downstream positions (four tested) does not dimerize despite the inclusion of the whole of a previously proposed dimerization domain (nucleotides 295-401); (2) an RNA starting between nucleotides 242 and 249 (five positions tested) dimerizes to a variable extent depending on the starting position; (3) an RNA starting at nucleotide 233 or at other upstream positions (five tested) is fully or > 80% dimeric; (4) an RNA starting at nucleotide 1 but lacking the 233-251 or the 242-251 region is, respectively, fully monomeric or about 50% monomeric; (5) the 343-401 region contains two strings of G's (GGGGG367 and GGG384) that had been postulated to promote genome dimerization through the formation of guanine quartets. We have deleted the 379-401, 358-401, and 343-401 regions from otherwise dimeric RNAs without changing their ability to dimerize. We reach three conclusions: (1) a dimerization signal exists upstream of the major 5' splice donor (nucleotide 290); (2) the previously proposed downstream dimerization domain is insufficient to promote dimerization and has a 3' half that is not necessary to obtain fully dimeric RNAs; (3) the 5' boundary of the HIV-1 dimerization domain is located somewhere between nucleotides 233 and 242, and the 3' boundary is located no farther than at nucleotide 342, making it possible that the 5' and 3' boundaries of the HIV-1 dimerization domain are both located within the leader sequence. We speculate that the 248-270 or 233-285 region forms a hairpin that is the core dimerization domain of HIV-1 RNA.

Base Sequence↗

PubMed1994

Structural dynamics of translating ribosomes: 16S ribosomal RNA bases that may move twice during translocation.

Recent footprinting, sedimentation and neutron-scattering results obtained in vivo or on pre-translocation and post-translocation ribosomal complexes are integrated with cross-linking and immunoelectron microscopy information. It is proposed that the 30S subunit pulses during translocation and that its pre- and post-translocation structures are not necessarily identical. Accordingly, translocation is characterized by three consecutive conformational states of the 30S and 50S subunits. State 1 (the pre-translocation state) lasts until the elongation factor EF-G.GTP complex binds to the ribosome or adopts the GTPase conformation. State 2 (the translocation state, or the peak or plateau of the pulse) follows and lasts until EF-G adopts a subsequent conformation or is released from the ribosome. State 3 (the post-translocation state) ensues and lasts until A (aminoacyl) site binding of aminoacyl-tRNA. In state 2, 16S RNA hairpins 26 and 33-33A, located in the platform and the head of the 30S subunit, respectively, become kinked or twisted, and residue A1503, near the decoding site, becomes exposed. A platform twist is associated with P (peptide) to E (exit) site tRNA movements and a head twist with pivoting of the peptidyl-tRNA elbow from the A to the P site, around a (retractable?) S19 domain. These twists result in an unlocking of the platform and the head from the 50S subunit. Exposure of A1503 is tentatively associated with movements of mRNA or tRNA anticodon stem-loops. These twisted or otherwise-exposed residues readopt their previous setting upon completion of translocation, i.e. states 1 and 3 of 16S RNA differ more from state 2 than from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗

PubMed1992

In vivo chemical footprinting of the Escherichia coli ribosome.

We have studied the in vivo chemical accessibility of 16S rRNA residues A349-G1505 in the small subunit of the Escherichia coli ribosome. Exponentially growing E. coli cultures were reacted with dimethyl sulfate, and the reactive sites on the 16S ribosomal RNA were analyzed by reverse transcription, an assay which detects reactions at N1-A and N3-C. In agreement with previous in vitro results, three regions of 16S RNA appeared particularly reactive to dimethyl sulfate: hairpin 27 (residues A892-A915) of the central domain, and hairpin 33-33A (residues A994-C1037) and the tip of hairpin 41 (residues A1256-A1275), both from the 3' major domain. These three regions contained 52% of the reactive residues but only 8% of the residues scanned. In contrast to previous in vitro results, three small sections of 16S RNA appeared protected: the tip of hairpins 26 (residue A845) and 31 (residues A968-A969), and residues A1418, A1441, and A1483 of the middle body of hairpin 44. Four of the dimethyl sulfate reactive sites (A831, C948, A1019, and C1192) are located in positions usually assumed to be double-stranded (helices 26, 30, 33-33A, and 34), which suggests alternative structures for these helices at least during part of the translation process, as if the residues in question belonged to "conformational switches." The addition of chloramphenicol protected residues A831, A1035-A1036, and A1503, which suggests that they belong to the mobile regions of the elongating ribosome, and become exposed during some transition(s) from one ribosomal state to the other during the elongation cycle.

Adenosine↗

PubMed1991

Interaction of ribosomal protein S1 and initiation factor IF3 with the 3' major domain and the decoding site of the 30S subunit of Escherichia coli.

We have studied the effect of the binding of ribosomal protein S1 and initiation factor IF3 on the accessibility of nucleotide residues 584-1506 in the small subunit of the Escherichia coli ribosome. Protein S1 strongly decreases RNase V1 attack at G1164, in hairpin 40 of the 3' major domain, and weakly decreases DMS attack at C1302, in the central loop of the 3' major domain, and at A1503, in the 3' minor domain. It also weakly increases the DMS reactivity of A1004, in the 3' major domain, and of A901, in the central domain. Factor IF3 strongly decreases RNase V1 attack (but not dimethyl sulfate attack) at A1408, in the decoding site, and weakly protects A1500, in the 3' minor domain and near the colicin E3 cleavage site. Neomycin does not interfere with this effect of IF3, but IF3 interferes with the protective effect of neomycin against dimethyl sulfate attack at A1408.

Base Sequence↗

PubMed1990

Delta Lys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12.

Five-residue-long deletions centered on Ala63, Ala75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118-Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12 delta Lys120. L7/L12 delta Lys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28-35% ethanol in 0.25-0.5 M NH4Cl. It follows that delta Lys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.

Amino Acid Sequence↗