Phosphorylation of Escherichia coli translation initiation factors by the bacteriophage T7 protein kinase.

The lytic coliphage T7 encodes a serine/threonine-specific protein kinase which supports viral reproduction under suboptimal growth conditions. Expression of the protein kinase in T7-infected Escherichia coli cells results in the phosphorylation of 30S ribosomal subunit protein S1, and initiation factors IF1, IF2, and IF3, as determined by high-resolution two-dimensional gel electrophoresis and specific immunoprecipitation analysis. Phosphorylation occurs either exclusively on threonine (IF1, IF3, S1) or on serine and threonine (IF2). There is no phosphorylation of these proteins in uninfected cells or in cells infected with T7 lacking the protein kinase function. Phosphorylation of the initiation factors coincides with the onset of T7 late protein synthesis, occurring 9-12-min postinfection. T7 late protein synthesis, otherwise inhibited in ColIb plasmid-containing cells, is specifically supported by expression of the protein kinase. These results provide the first evidence for the functional involvement of protein phosphorylation in the control of bacterial translation.     

The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli.

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).     

Both the 5'' untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro.

The role of RNA sequences in the 5' leader region between the cap site and initiating AUG in mediating translation was examined in vitro. Hybrid mRNAs were synthesized in which the cognate leader sequence was replaced with either optimized or compromised leader sequences, and translational efficiency was measured for six different coding regions. Translation was most efficient with a leader containing the 5' untranslated region from Xenopus beta-globin and an optimized initiation sequence. Compared with the cognate leaders, this hybrid was observed to increase translation of the various coding regions as much as 300-fold. The translational efficiencies of the different coding regions also varied substantially. In contrast to earlier suggestions that increased leader efficiency results from higher affinity of the leader for a limiting factor, our experiments suggest that increased translation from the beta-globin hybrid leader sequence results from more rapid initiation of translation.     

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

An NMR characterization of the regA protein-binding site of bacteriophage T4 gene 44 mRNA

The conformations of two RNA dodecamers that differ markedly in affinity for the regA protein from bacteriophage T4 have been examined by NMR to see if the ability of that protein to discriminate between mRNAs is based on pre-existing differences in their three-dimensional structures. One of the RNAs examined has the same sequence as the site where regA protein binds when it inhibits the expression of gene 44's mRNA. The second RNA differs from the first in having a U instead of a G at position -9; it binds regA protein 100 times less tightly. The NMR data indicate that both RNAs have similar single-stranded conformations and that they each resemble an isolated strand of a double helix. They also show that most, if not all of the ribose rings in both molecules have appreciable 2'-endo puckering. It is unlikely that regA protein distinguishes between these two molecules on the basis of differences in their global conformations in solution.     

A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli

We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."     

The dependence of the E.coli gene expression level on the structure of the translation initiation region (TIR). IV. Distal complementary TIR interactions with the mRNA coding region

To evaluate the effect on translation of distal regions of the encoding mRNA part capable of the complementary binding to the ribosome binding site (RBS), a series of plasmids were constructed containing fragments inserted into the il3 gene and determining secondary interactions in mRNA. A comparison of the levels of the in vivo gene expression showed that the complementary interactions of the translation initiation region (TIR) with distal regions of the mRNA encoding part affect translation. The effectiveness of these interactions decreased with an increase in the distance between the RBS and the complementary mRNA region, whereas the secondary structure formed by the TIR and the adjacent mRNA region was more stable despite the presence of regions in mRNA capable of forming energetically more favorable structures involving these elements.     

More than 150 nucleotides flanking the initiation codon contribute to the efficiency of the ribosomal binding site from bacteriophage T7 gene 1.

The ribosomal binding site (RBS) from gene 1 of bacteriophage T7 was isolated on fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene to control the translation of its sequence. A 29 base pair sequence containing all elements generally believed to be essential for the RBS's showed extremely low activity. Additional upstream and downstream sequences were required to obtain a several orders of magnitude higher efficiency. By contrast, areas further downstream than +112 nucleotides from the initiator proved to be inhibitory, whereas the presence of an upstream RNaseIII cleavage site showed a strong stimulatory effect. This suggests that tertiary structures are involved in the function of the RBS studied. The efficient RBS's were complexed by ribosomes at much lower concentrations of the mRNA than the weak ones.     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.