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J M Zengel

Publications and source records attributed to J M Zengel.

At least 37 records · Page 2Linked to original sources
PubMed1990

Transcriptional organization of the S10, spc and alpha operons of Escherichia coli.

We have investigated the transcription patterns at the inter-operon regions between the S10 and spc, and spc and alpha ribosomal protein operons of Escherichia coli. Newly synthesized transcripts were characterized by RNase T1 protection experiments, and accumulated transcripts were mapped with S1 nuclease. With both techniques we found that about 75% of the RNA polymerases transcribing the S10 operon terminated at the position of a typical rho-independent terminator. In contrast, most or all RNA polymerases transcribing the spc operon continued into the alpha operon. Nevertheless, we observed that about 30% of the transcripts of the alpha operon were initiated at the alpha operon promoter.

Base Sequence↗
PubMed1990

Escherichia coli ribosomal protein L4 stimulates transcription termination at a specific site in the leader of the S10 operon independent of L4-mediated inhibition of translation.

Transcription of the 11-gene S10 operon of Escherichia coli is inhibited by excess ribosomal protein L4, the product of the third gene of the operon. Previous studies suggested that L4 regulates transcription by modulating the level of readthrough at an attenuator in the S10 leader. To understand better the molecular details of the transcriptional regulation, we have determined the site of L4-induced termination of transcription using a method that allows us to map the 5' and 3' ends of newly synthesized RNA. Our results indicate that L4 stimulates termination about 140 bases from the transcription start site. Thus, the termination point is more than 30 bases upstream from the most proximal structural gene of the S10 operon, and coincides with a string of U residues on the descending side of a terminator-like hairpin structure. Since L4 is also known to inhibit translation of the S10 operon, we have analyzed the role of translation control in the protein's regulation of transcription by deleting sequences downstream from the termination site, including bases involved in translation initiation of the proximal structural gene. We find that the first 150 bases of the S10 leader contain the information sufficient for L4-mediated attenuation control and, therefore, that L4 regulates transcription by a mechanisms that is independent of the protein's inhibition of translation.

Base Sequence↗
PubMed1990

Ribosomal protein L4 stimulates in vitro termination of transcription at a NusA-dependent terminator in the S10 operon leader.

The 11-gene S10 ribosomal protein operon of Escherichia coli is under the autogenous control of L4, the product of the third gene of the operon. Ribosomal protein L4 inhibits both transcription and translation of the operon. Our in vivo studies indicated that L4 regulates transcription by causing premature termination within the untranslated S10 operon leader. We have now used an in vitro transcription system to study the effect of purified L4 on expression of the S10 operon. We find that the cell-free system reproduces the in vivo observations. Namely, in the absence of L4, most of the RNA polymerases read through the termination site in the S10 attenuator; the addition of L4 results in increased termination at this site. However, RNA polymerase does not terminate at the S10 attenuator, with or without L4, unless an additional factor, protein NusA, is added to the transcription reaction. These results suggest that the attenuator in the S10 operon is a NusA-dependent terminator whose efficiency is regulated by ribosomal protein L4.

Bacterial Proteins↗
PubMed1990

Autogenous control is not sufficient to ensure steady-state growth rate-dependent regulation of the S10 ribosomal protein operon of Escherichia coli.

The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ. The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon. Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did. However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth. Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control. These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control.

Bacterial Proteins↗
PubMed1989

Translational coupling of the two proximal genes in the S10 ribosomal protein operon of Escherichia coli.

We have examined the translational coupling between the first two genes in the S10 ribosomal protein operon. We isolated mutations blocking the translation of the first gene of the operon, coding for S10, and monitored their effects on translation of the downstream gene, coding for L3. All of the mutations inhibiting S10 synthesis also affected the synthesis of L3. However, these experiments were complicated by decreased mRNA synthesis resulting from transcription polarity, which we could only partially eliminate by using a rho-100 strain. To completely eliminate the problem of transcription polarity and obtain a more accurate measurement of the coupling, we replaced the natural S10 promoter with a promoter used by the bacteriophage T7 RNA polymerase. As expected, the T7 RNA polymerase was not subject to transcription polarity. Using this system, we were able to show that a complete abolishment of S10 translation resulted in an 80% inhibition of L3 synthesis. Other experiments show that the synthesis of L3 goes up as a function of increasing S10 synthesis, but the translational coupling does not assure strictly proportional output from the two genes.

DNA Mutational Analysis↗
PubMed1988

Secondary structure of the leader transcript from the Escherichia coli S10 ribosomal protein operon.

Genetic analysis of the autogenous control of the S10 ribosomal protein operon of Escherichia coli has suggested that the secondary or tertiary structure of the leader transcript is important for this regulation. We have therefore determined the secondary structure of the leader by enzyme digestion and chemical modification. Our results suggest that the 172 base leader exists in two forms, differing only immediately upstream of the Shine-Dalgarno sequence of the first gene. We discuss the possibility that the equilibrium between these alternate structures is important for the L4-mediated regulation of translation of the S10 operon. We have also determined the structure of several mutant transcripts. Correlation of these structures with the regulatory phenotypes suggest that a hairpin about 50 bases upstream of the first gene is essential for the control of translation of the operon. Finally, our results show that a two base substitution in an eight base loop destabilizes the attached stem.

Base Sequence↗
PubMed1987

Autogenous control of the S10 ribosomal protein operon of Escherichia coli: genetic dissection of transcriptional and posttranscriptional regulation.

The S10 ribosomal protein operon is regulated autogenously by the product of one of the genes of the operon, the gene encoding ribosomal protein L4. We have used site-directed mutagenesis to isolate leader mutations affecting L4 control. The phenotypes of these mutants demonstrate that L4 regulates both transcription and translation of the S10 operon. Several mutations abolish both levels of L4 control; others eliminate either transcriptional or translational control with little or no effect on the other mode of regulation. We conclude that L4-mediated transcriptional and translational control share some sequence requirements, but the two regulatory processes recognize somewhat different features of the S10 leader. Primary as well as secondary structures within the S10 leader appear to be involved.

Escherichia coli↗
PubMed1986

Precursor for elongation factor Tu from Escherichia coli.

The tufA gene, one of two genes in Escherichia coli encoding elongation factor Tu (EF-Tu), was cloned into a ColE1-derived plasmid downstream of the lac promoter-operator. In cells carrying this plasmid, the synthesis of EF-Tu was increased four- to fivefold upon the addition of isopropyl-beta-D-thiogalactopyranoside (an inducer of the lac promoter). This condition led to the synthesis of a novel protein, called pTu, which comigrated with EF-Tu on a sodium dodecyl sulfate-polyacrylamide gel but could be separated on an isoelectric focusing gel, since pTu is slightly more basic than EF-Tu. The synthesis of pTu could also be induced by the synthesis of a hybrid protein containing just the amino-terminal half of the EF-Tu protein. Genetic data suggest that pTu is the product of the tufA and tufB genes. The pTu protein was shown to be related to EF-Tu by gel electrophoresis of tryptic peptides. Pulse-chase experiments suggest that pTu is a precursor of EF-Tu. Interestingly, in a classic membrane fractionation procedure, EF-Tu was found in the cytosolic fraction, whereas pTu was partitioned with the outer membrane.

Bacterial Proteins↗
PubMed1986

Transcription of ribosomal genes during a nutritional shift-up of Escherichia coli.

We measured the differential transcription rates of individual ribosomal operons after a nutritional shift-up. All operons showed a transient increase in transcription. However, the response of the S10 ribosomal protein operon was much stronger than that of any other operon. We propose that only the S10 operon is autogenously regulated by a transcription attenuation mechanism.

Bacterial Proteins↗
PubMed1985

Genetic dissection of stringent control and nutritional shift-up response of the Escherichia coli S10 ribosomal protein operon.

The S10 operon of Escherichia coli is autogenously regulated by L4, one of 11 ribosomal proteins encoded by the operon. We have previously shown that L4 regulates transcription of the operon by modulating the level of read-through at an attenuator in the S10 leader. To determine the physiological roles of both L4-mediated attenuation and the regulation of transcription initiation, we have constructed mutations eliminating their two regulatory targets, the S10 leader and the S10 promoter. Our results indicate that stringent control requires only the S10 promoter and therefore is mediated at the level of initiation. However, growth-medium-dependent control after a nutritional shift-up involves regulation of both initiation of transcription at the promoter and transcription read-through at the attenuator.

Bacterial Proteins↗
PubMed1985

Nucleotide sequence of the alpha ribosomal protein operon of Escherichia coli.

In Escherichia coli some 19 transcription units encoding the 52 ribosomal proteins are scattered throughout the genome. One of the units, the alpha operon, encodes genes for the ribosomal proteins S13, S11, S4 and L17 as well as the alpha subunit of RNA polymerase. We report here the complete 3.0 kb nucleotide sequence of the alpha operon. In addition, we have determined by S1 nuclease mapping the site of transcription termination in this operon.

Amino Acid Sequence↗
PubMed1985

Transcriptional control of the S10 ribosomal protein operon of Escherichia coli after a shift to higher temperature.

In the 5 to 10 min immediately following a shift from 30 to 42 degrees C, the differential synthesis rates of ribosomal proteins encoded by the 11-gene S10 operon are transiently decreased. This effect results largely from a two- to threefold decrease in the differential rate of transcription of the operon. The inhibition of mRNA synthesis is apparently due to two types of control: (i) initiation of transcription at the S10 promoter is inhibited and (ii) readthrough at the attenuator in the S10 leader is decreased. Both of these effects on transcription are independent of the heat shock regulatory gene, htpR. Furthermore, the inhibition of transcription is observed in both relA+ and relA cells, suggesting that the temperature-induced repression does not require the relA-dependent accumulation of guanosine tetraphosphate (ppGpp). However, recovery from the heat shock was slower in relA+ strains than in relA strains. None of the other ribosomal protein operons that we analyzed showed such a strong decrease in transcription after the heat shock.

Escherichia coli↗
PubMed1984

The nucleotide sequence of the Escherichia coli fus gene, coding for elongation factor G.

We have determined the nucleotide sequence of the Escherichia coli fus gene, which codes for elongation factor G. The protein product of the sequenced gene contains 703 amino acids, with a predicted molecular weight of 77,444. The fus gene shows the nonrandom pattern of codon usage typical of ribosomal proteins and other proteins synthesized at a high level. We have identified several potential promoter sequences within the gene. One of these sequences may correspond to the secondary promoter for expression of the downstream tufA gene (encoding elongation factor Tu) whose activity has been described previously (1,2). A comparison of the nucleotide and amino acid sequences of elongation factors G and Tu reveals a limited but significant homology between the two proteins within the 150 amino acid residues at their amino-terminal ends.

Amino Acid Sequence↗
PubMed1984

Role of attenuation in growth rate-dependent regulation of the S10 r-protein operon of E. coli.

We have investigated the transcription of the 11 gene S10 ribosomal protein operon of Escherichia coli under various growth conditions. The differential synthesis rate of structural gene message increases 2- to 2.5-fold immediately after a shift-up from glycerol minimal medium to glucose plus amino acids. After the initial increase, the transcription rate goes through several oscillations before reaching the new steady-state rate. By comparing the rates of transcription of leader and structural genes, we conclude that these oscillations are due predominantly to changes in the level of read-through at the S10 attenuator. This regulation of attenuation can account for most of the variations in protein synthesis from the S10 operon after a shift. We also measured the level of read-through in cells growing exponentially in different growth media. Over a 2.5-fold range in growth rates, the read-through changed less than 50%. Thus, regulation of attenuation cannot explain the growth-dependent regulation of ribosomal protein synthesis during steady-state growth. Apparently, additional mechanisms are required to control the expression of the S10 operon in exponentially growing cells.

Escherichia coli↗
PubMed1983

Transcription of the S10 ribosomal protein operon is regulated by an attenuator in the leader.

Previous studies have shown that ribosomal protein L4 specifically inhibits the expression of its own operon, the 11-gene S10 operon. To elucidate the mechanism for this regulation, we have examined the effect of protein L4 on transcription of the S10 operon. Hybridization and gel electrophoresis studies indicate that in the presence of excess L4 only RNA molecules about 140 bases long are transcribed from the S10 operon. These short RNA molecules contain the leader, but not structural gene, sequences. Our results suggest that protein L4 stimulates premature termination (attenuation) of transcription about 30 bases upstream from the start of the first structural gene of the S10 operon. The attenuation appears to be independent of the regulation of translation of the operon. We suggest that attenuation of transcription plays a primary role in the autogenous regulation of the S10 operon.

Escherichia coli↗
PubMed1982

A secondary promoter for elongation factor Tu synthesis in the str ribosomal protein operon of Escherichia coli.

The str operon of Escherichia coli contains genes for ribosomal proteins S12 and S7 and for elongation factors EF-G and EF-Tu (Jaskunas et al. 1975). We have subcloned various segments of DNA from this operon onto multicopy plasmids. We found that cells carrying a recombinant plasmid which lacks the major promoter for the str operon but contains the 5' portion of the EF-Tu gene synthesize a novel protein which we have identified as a truncated EF-Tu molecule. Moreover, cells carrying plasmids with an intact EF-Tu gene synthesize the elongation factor at a 3- to 5-fold higher rate than haploid cells. Thus the EF-Tu gene can be expressed in the absence of the major promoter for the str operon. This expression is not due to read-through from plasmid promoters, but it is dependent on the presence of the distal portion of the EF-G gene on the plasmids. These results indicate that there is a secondary promoter for EF-Tu expression, apparently located within the structural gene for elongation factor EF-G.

Cloning, Molecular↗