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PubMed2000

The human and mouse methylenetetrahydrofolate reductase (MTHFR) genes: genomic organization, mRNA structure and linkage to the CLCN6 gene.

Methylenetetrahydrofolate reductase (MTHFR), a pivotal enzyme in folate metabolism, regulates the proportional distribution of one-carbon moieties between cellular methylation reactions and nucleic acid synthesis. The organization of the MTHFR gene and the structure of its mRNA were characterized in human and mouse. There are three mRNA transcripts of 2.8, 7.2 and 9.8 kb in human and two of 3.2 and 7.5 kb in mouse. Northern blot analysis revealed that human MTHFR MRNA is only present at low abundance in most tissues tested. Five kilobases of sequence flanking the 3' end of the human gene were isolated, and polyadenylation sites were defined by 3' RACE. The shorter 2.8 kb transcript and the two larger 7.2 and 9.8 kb transcripts utilize different polyadenylation signal sequences, 629 and 4937 bp downstream of the stop codon, respectively. The two mRNA species in mouse also result from differential polyadenylation. Approximately 7 and 3.5 kb upstream of the human and mouse genes, respectively, were isolated and sequenced. Transcription start sites in human MTHFR were mapped using 5' RACE. The 2.8 and 7.2 kb mRNAs originate from one of two transcription start sites that are 206 and 243 bp upstream of the ATG initiation codon, whereas transcription of the 9.8 kb mRNA is initiated at a start site located 2.8 kb upstream of the translation start codon. The putative MTHFR promoter does not have a TATA box but contains CpG islands and multiple potential Sp1 binding sites. The MTHFR gene was finely mapped to interval 16 of chromosome 1p36.3, a region deleted in many tumors, by establishing a close linkage to CLCN6, a putative chloride channel gene. A novel CA-repeat polymorphism identified within intron 2 of the CLCN6 gene may be useful in assessing loss of heterozygosity in such tumors. The multiple MTHFR mRNA species identified in this report may reflect an underlying complex set of gene regulatory mechanisms acting through an alternative transcription start site and/or polyadenylation signal sequence utilization.

3' Untranslated Regions↗

PubMed1998

Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus.

Inoculation of Vigna unguiculata (cowpea) with transcripts synthesized in vitro from a genome-length cDNA clone of the cowpea strain of southern bean mosaic virus (SBMV-C) resulted in a systemic SBMV-C infection of this host. Capped RNA was about five times more infectious than uncapped RNA as determined by a local lesion assay. The SBMV-C cDNA clone was also used for mutagenesis of the four SBMV-C open reading frames (ORFs). ORF1, ORF3, and coat protein (CP) mutants were not infectious in cowpea. Electroporation of cowpea protoplasts with mutant transcripts demonstrated that the ORF1, ORF3, and CP gene products were not required for SBMV-C RNA synthesis, and the ORF1 and ORF3 gene products were not required for SBMV-C assembly. From these results, it was concluded that the ORF1 and ORF3 proteins and the CP are required for SBMV-C cell-to-cell movement. One of the ORF3 mutants pSBMV2-UAA1833 contained a nonsense codon between the predicted -1 ribosomal frameshift site (SBMV-C nucleotides 1796-1802) and a potential ORF3 translation initiation codon at SBMV-C nucleotide 1895. The lack of infectivity of this mutant suggested that ORF3 was expressed by a -1 ribosomal frameshift in ORF2 rather than by initiation of translation at nucleotide 1895.

Electroporation↗

PubMed1999

Functional analysis of a mutation occurring between the two in-frame AUG codons of human angiotensinogen.

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1987

Translation framing code and frame-monitoring mechanism as suggested by the analysis of mRNA and 16 S rRNA nucleotide sequences.

Protein coding sequences carry an additional message in the form of a universal three-base periodical pattern (G-non-G-N)n, which is expressed as a strong preference for guanines in the first positions of the codons in mRNA and lack of guanines in the second positions. This periodicity appears immediately after the initiation codon and is maintained along the mRNA as far as the termination triplet, where it disappears abruptly. Known cases of ribosome slippage during translation (leaky frameshifts, out-of-frame gene fusion) are analyzed. At the sites of the slippage the G-periodical pattern is found to be interrupted. It reappears downstream from the slippage sites, in a new frame that corresponds to the new translation frame. This suggests that the (G-non-G-N)n pattern in the mRNA may be responsible for monitoring the correct reading frame during translation. Several sites with complementary C-periodical structure are found in the Escherichia coli 16 S rRNA sequence. Only three of them are exposed to various interactions at the surface of the small ribosomal subunit: (517)gcCagCagCegC, (1395)caCacCgcC and (1531)auCacCucC. A model of a frame-monitoring mechanism is suggested based on the weak complementarity of G-periodical mRNA to the C-periodical sites in the ribosomal RNA. The model is strongly supported by the fact that the hypothetical frame-monitoring sites in the 16 S rRNA that are derived from the nucleotide sequence analysis are also the only sites known to be actually involved or implicated in rRNA-mRNA interactions.

Base Sequence↗

PubMed1990

Translational autocontrol of the Escherichia coli ribosomal protein S15.

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Base Composition↗

PubMedDate not supplied

Structure and function of the yeast URA3 gene: expression in Escherichia coli.

The expression of the cloned Saccharomyces cerevisiae URA3 gene in Escherichia coli on both plasmid and phage vectors was studied. Isolates of the gene from two different laboratory strains of yeast differ in their ability to be expressed in E. coli in the absence of external adjacent promoters of transcription. The DNA sequence of the two genes was determined and revealed several differences in the DNA flanking the structural gene. One base change alters the "Pribnow-box" of an E. coli promoter present in the yeast sequences. Three amber alleles of the yeast gene were also cloned from yeast. Two of the alleles could be suppressed in E. coli by a tRNA suppressor mutation. One of the amber alleles was determined to be a mutation in the seventh codon of the structural gene, thereby establishing the reading frame and extent of the coding sequence. The initiator codon of the reading frame encoding the URA3 structural gene is preceded by two other ATG codons in a different reading frame 61 and 79 bp away. The nearer ATG begins an open reading frame that overlaps the structural gene sequences by 17 bp. With the DNA sequence of the URA3 gene many of the common yeast vector plasmids are now completely known at the level of DNA sequence.

Amino Acid Sequence↗

PubMed1994

A new look at the promoter of the human monoamine oxidase A gene: mapping transcription initiation sites and capacity to drive luciferase expression.

Monoamine oxidase (MAO) A (EC 1.4.3.4) oxidizes norepinephrine and serotonin and is expressed in a cell type-specific manner. Recent evidence that MAO A-deficient males in a large Dutch kindred suffer from mild mental retardation and occasional episodes of impulsive aggressive behavior makes it important to understand how the human MAO A promoter is regulated. Conventional primer extension analyses of MAO A mRNA in earlier studies predicted incorrect transcription initiation sites for the human MAO A promoter. Reverse transcription and polymerase chain reaction (PCR) readily detected MAO A mRNA initiated 5' to -135 bp but not 5' to -226 bp (5' to the ATG initiation codon). PCR-assisted primer extension and RNase protection assays reveal that most MAO A mRNA is initiated between -30 and -40, which resembles a eukaryotic initiator element. Depending on the tissue source, a minor, variable proportion of MAO A mRNAs is initiated more distally at approximately -95 and -136, within the more proximal of two 90-bp GC-rich tandem repeats. Genomic DNA segments spanning -4 to -200 and -465 or -935, but not -4 to -82, drive robust luciferase expression in mammalian cells. We conclude that (a) the primary transcription initiation site occurs at a putative initiator (lnr) element located between -30 and -40, with a minor, tissue-specific proportion of additional initiation near -95 and -136; and (b) MAO A-luciferase reporter constructs that contained all the known transcription initiation sites exhibited no evidence for inhibitory cis elements between -200 and at least -935. The apparent inhibitory activity previously reported for sequences 5' to the most proximal PvuII site may have resulted from the use of partial promoter constructs that omitted the putative lnr element.

Base Sequence↗

PubMed1995

Functional characterization of the promoter region of the platelet-activating factor receptor gene. Identification of an initiator element essential for gene expression in myeloid cells.

To understand the molecular mechanisms that direct the expression of the gene encoding the platelet-activating factor (PAF) receptor, the 5'-flanking region of the human PAF receptor gene was cloned, and its promoter activity in myeloid cell lines was characterized. By the 5'-rapid amplification of cDNA ends method and primer extension, the transcription initiation site was mapped to an adenosine residue 137 bases upstream of the ATG translation initiation codon. The promoter region lacks a typical TATA or CCAAT box. However, the sequence encompassing the transcription initiation site shows high homology to the initiator (Inr) sequence. Transfection of promonocytic U937 cells with recombinant plasmids containing a series of truncated segments of the 5'-flanking region linked to the luciferase reporter gene revealed that the sequence from nucleotides -44 to +27 relative to the transcription initiation site was sufficient to promote a high level of gene expression. The promoter activity was much lower in nonexpressing HeLa cells and promyelocytic HL-60 cells, which express relatively low levels of the PAF receptor. Gel mobility shift analysis demonstrated the binding of nuclear factors extracted from myelocytic cells to the -16/+18 sequence containing the Inr element. No binding activity was detected using the nuclear extracts from the nonmyelocytic HeLa cells. The DNA-protein complexes were sequence-specific since the binding was not significantly affected by the mutated Inr sequences or the Inr sequence of the terminal deoxynucleotidyltransferase gene. Furthermore, point mutations in the Inr element significantly reduced promoter activity in both U937 and THP-1 cell lines. When Me2SO or retinoic acid was used to induce granulocytic differentiation of HL-60 cells, a distinct Inr-protein complex was induced concurrently, but the complex was not observed in 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiated HL-60 cells or Me2SO-induced differentiated U937 cells, indicating that the inducible Inr binding activity is granulocyte-specific. These results suggest that distinct nuclear factors interact with the unique Inr element and play a role in the transcriptional regulation of the PAF receptor in various myeloid cells.

Base Sequence↗

PubMed1998

Human leukotriene B4 omega-hydroxylase (CYP4F3) gene: molecular cloning and chromosomal localization.

Leukotriene B4 (LTB4) omega-hydroxylase catalyzes the conversion of LTB4 into a biologically less active product, 20-hydroxy-LTB4. In a preceding paper (Kikuta et al., 1993), we showed human polymorphonuclear leukocyte (PMN) LTB4 omega-hydroxylase to be a novel form of cytochrome P450, designated CYP4F3, on the basis of its cDNA cloning and expression in yeast cells. Here, we have isolated the gene encoding CYP4F3 and determined its genomic organization and chromosomal localization. The CYP4F3 gene contained 13 exons and spanned approximately 22.2 kb. The cDNA of CYP4F3 contained 5050 nucleotides excluding the poly(A) tail. The translation initiation codon (ATG) was present in exon II. Primer extension and S1 mapping analyses indicated that the transcription initiation site is 49 nucleotides upstream from the 3' end of exon I, and no other initiation sites were detected. A TATA-box-like sequence (TACAT) and 120-b GC-rich sequence were observed just before transcription initiation site. Several putative regulating elements recognized by the GATA family, MZF-1, CACCC binding protein, and C/EBP, were identified in its 5' flanking region. Genomic DNA screening for CYP4F3 and Southern blot analysis suggested the existence of other CYP4F genes in addition to CYP4F3 and CYP4F2 in the human genome. Fluorescence in situ hybridization demonstrated that the CYP4F3 gene is located at 19p13.2.

Amino Acid Sequence↗

PubMed1989

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101.

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.

Amino Acid Sequence↗

PubMed2005

Repression of human gamma-globin gene expression by a short isoform of the NF-E4 protein is associated with loss of NF-E2 and RNA polymerase II recruitment to the promoter.

Binding of the stage selector protein (SSP) to the stage selector element (SSE) in the human gamma-globin promoter contributes to the preferential expression of the gamma-gene in fetal erythroid cells. The SSP contains the transcription factor CP2 and an erythroid-specific partner, NF-E4. The NF-E4 gene encodes a 22-kDa polypeptide employing a non-AUG initiation codon. Antisera specific to NF-E4 detects this species and an additional 14 kDa protein, which initiates from an internal methionine. Enforced expression of p14 NF-E4 in the K562 fetal/erythroid cell line, and in primary erythroid cord blood progenitors, results in repression of gamma-gene expression. Biochemical studies reveal that p14 NF-E4 interacts with CP2, resulting in diminished association of CP2 with the SSE in chromatin immunoprecipitation assays. p45 NF-E2 recruitment to the gamma-promoter is also lost, resulting in a reduction in RNA polymerase II and TBP binding and a fall in promoter transcriptional activity. This effect is specific, as enforced expression of a mutant form of p14 NF-E4, which fails to interact with CP2, also fails to repress gamma-gene expression in K562 cells. These findings provide one potential mechanism that could contribute to the autonomous silencing of the human gamma-genes in adult erythroid cells.

Erythroid Precursor Cells↗

PubMed1996

Characterization of the regulatory regions of murine alpha 2C2 adrenoceptor subtype gene.

In order to delineate the regulatory mechanisms underlying the control of alpha 2 adrenoceptor expression, the sequence of 5' (1145 bp) and 3' (2682 bp) flanking regions of murine alpha 2C2 subtype gene were determined and characterized from a genomic phage clone MA2C2. The 5' flanking region has no TATA box yet with high GC content. The 3' flanking region is marked by the presence of a polyadenylation signal 2.3 kb down stream from the stop codon. The transcription start site was mapped by 5' rapid amplification of cDNA ends (RACE) and primer extension assays at nucleotide A, 415 bp upstream from the first initiation codon and resides in a motif resembling the consensus sequence of initiator found in many TATA-less promoters. An NcoI fragment (4.7 kb) immediately upstream from the translation initiation site was linked to a reporter gene lacZ. Using an in vitro transfection assay, cell lines of renal or neural origin were identified as permissive hosts for alpha 2C2 subtype expression. With its core promoter clearly defined and sequence of the regulatory regions at hand, this in vitro gene transfer system will facilitate the identification of putative cis-elements and transcription factors key to alpha 2C2 adrenoceptor expression.

Animals↗

PubMed1982

Relationship between structure of the 5' noncoding region of viral mRNA and efficiency in the initiation step of protein synthesis in a eukaryotic system.

To determine whether the rate of protein synthesis is controlled by the structure of mRNA near its 5' terminus, protein-synthesizing ability, especially in its initial stage, was compared among RNAs of plant viruses. Those viruses used here contain several definite pieces of single-stranded RNA. Each of these RNAs acts as a messenger. Cucumber mosaic virus (CMV) RNA 5 synthesizes a small amount of a protein, Mr 7000, in an in vitro protein-synthesizing system from wheat germ or reticulocyte. Brome mosaic virus (BMV) RNA 4 synthesizes a large amount of a coat protein under the same conditions. Both RNAs have the same 5'-cap structure and a short noncoding region (10 nucleotides in CMV RNA 5 and 9 in BMV RNA 4) between the 5' terminus and the initiation codon AUG. A sequence complementary to the 3' terminal of 18S ribosomal RNA is contained in BMV RNA 4 but is not apparent in CMV RNA 5. Formation of the initiation complex for protein synthesis by the 5'-terminal-labeled mRNA of cytoplasmic polyhedrosis virus was inhibited by the addition of unlabeled BMV RNA 4 whereas it was only slightly inhibited by unlabeled CMV RNA 5. BMV RNA 4, which has a sequence complementary to rRNA, can form the initiation complex more easily than CMV RNA5. It is concluded that an apparent complementary sequence in the 3' terminal of 18S rRNA in the 5' noncoding region of eukaryotic mRNA and the 5'-cap structure enhance the rate of initiation complex formation in protein synthesis.

Base Sequence↗

PubMed1989

Translation initiation at non-AUG triplets in mammalian cells.

In a previous report it was shown that mammalian ribosomes were capable of initiating translation at a non-AUG triplet when the initiation codon of mouse dihydrofolate reductase (dhfr) was mutated to ACG (Peabody, D.S. (1987) J. Biol. Chem. 262, 11847-11851). In order to assess the capacity of the mammalian translation apparatus to initiate at other non-AUG triplets, the initiator AUG of dihydrofolate reductase was converted to GUG, UUG, CUG, AGG, AAG, AUA, AUC, and AUU. These represent (with ACG) all the possible triplets that differ from AUG by only one nucleotide. The ability of each mutant to produce dihydrofolate reductase was assessed by in vitro transcription/translation of the mutant dhfr sequences under control of the bacteriophage SP6 promoter. Each of the triplets (with the exceptions of AGG and AAG) was able to direct the synthesis of apparently normal dihydrofolate reductase. Incorporation of [35S]tRNAifMet into the products of in vitro translation indicates that in each case the non-AUG triplet is able to direct initiation of the polypeptide chain with methionine. The mutant dhfr sequences were also inserted into the mammalian expression vector SVGT5 for expression in cultured monkey cells. The hierarchy of relative translation efficiencies was similar in vivo and in vitro.

Animals↗

PubMed1991

Dictyostelium discoideum essential myosin light chain: gene structure and characterization.

We have used a Dictyostelium essential myosin light chain (EMLC) cDNA clone to isolate additional cDNA clones which supply a different 3' sequence from that previously described. The revised cDNA sequence encodes a polypeptide of 150 amino acids. Amino acid residues 147-167 of the previously reported sequence are replaced by new residues 147 to 150. The new cDNA encodes a polypeptide with 66% amino acid sequence identity with the Physarum polycephalum EMLC, and approximately 30% identity with mammalian EMLC sequences. These new cDNA clones were used to isolate two genomic DNA fragments which contain the entire EMLC gene. The Dictyostelium EMLC gene contains a single intron located immediately 3' of the translation initiation codon and encodes a product most similar to MLC3 isoform of vertebrates. Primer extension analysis places the transcription initiation site approximately 90 nucleotides upstream of the translation initiation site. A DNA fragment containing 350 bases of sequence upstream of the putative transcription initiation site is sufficient to drive expression of a reporter gene upon reintroduction into growing Dictyostelium cells. In addition, the CAT reporter mRNA produced by this construct showed a pattern of developmental regulation similar to that previously reported for the endogenous EMLC mRNA. Based on comparison with published EMLC sequences from a variety of sources, the Dictyostelium EMLC shows slightly higher similarity to vertebrate EMLCs from striated muscle sources than nonmuscle sources. While Dictyostelium and human nonmuscle sequences display only 28% identity over their entire sequence, the region from residue 88 to 108 shows much higher identity (67%). The high evolutionary conservation of this region of the EMLC suggests it may play an important role in EMLC function, and as such, represents a good target for future mutagenesis studies.

Amino Acid Sequence↗

PubMed1997

In vitro mutational and inhibitory analysis of the cis-acting translational elements within the 5' untranslated region of coxsackievirus B3: potential targets for antiviral action of antisense oligomers.

The 5' untranslated region (5'UTR) of coxsackievirus B3 (CVB3) RNA forms a highly ordered secondary structure that has been implicated in controlling initiation of viral translation by internal ribosomal entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the 5'UTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron, the chloramphenicol acetyltransferase gene, designed to block ribosomal scanning, the CVB3 5'UTR was capable of directing the internal initiation of translation of the downstream reporter gene (P1), confirming the presence of an internal ribosomal entry site (IRES). This finding was further supported by the data on predicted secondary structures within the 5'UTR. Of special note, analysis of various deletion mutants demonstrated that the IRES of CVB3 is located roughly at stem-loops G, H, and I spanning nucleotides (nt) 529 and 630. The region from nt 1 to 63 (stem-loop A) also appears important, and it may be an essential binding site for translation initiation factors. Based on these findings, in vitro translation inhibition assays using RNA fragments of the 5'UTR as inhibitor were performed. Both antisense and sense RNA segments transcribed from these two cis-acting regions and the surrounding sequence of the initiation codon AUG showed strong inhibition of viral protein synthesis. Antisense molecules may inhibit translation by blocking ribosome and initiation factor binding within the 5'UTR via specific hybridization to their viral RNA target sequences, while sense sequences may function by competing with viral RNA for ribosomes and/or translation initiation factors. These cis-acting translational elements may serve as potential targets for the antiviral action of oligomers.

Animals↗

PubMed1994

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs.

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5-2-kDa difference between the M(r)s of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed.

Amino Acid Sequence↗

PubMed1991

Influence of mRNA determinants on translation initiation in Escherichia coli.

We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation.

Anticodon↗