A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin

The DNA sequence changes of 31 mutations altering the attenuation control mechanism of the histidine operon are presented. These mutations are discussed in terms of a model for operon regulation that involves a his leader peptide gene whose translation regulates formation of alternative stem-loop structures in the his leader messenger RNA. Three suppressible mutations generate nonsense codons (ochre and UGA) in the his leader peptide gene, demonstrating that translation of this gene is essential for operon expression. Eight mutations presumably reduce the efficiency of translation initiation of the his leader peptide gene, causing reduced levels of operon expression. Five of these mutations directly alter the leader peptide gene initiator codon (AUG). Three mutations alter sequences just in front of the initiator codon and presumably alter the ribosome recognition site. Fourteen mutations reduce the stability of the his leader mRNA stem-loop structures that are alternatives to the attenuator stem. The properties of these mutations provide support for the role of these stem-loop structures in preventing formation of the attenuator stem. Finally, we show that mutations that alter the attenuator stem suppress hisO mutations. This lends support to the proposal that these hisO mutations cause reduced levels of operon expression due to excessive attenuator stem formation. The properties of these 31 mutations provide substantial support for the model of his operon regulation described in this paper.     

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator

Two DNA fragments which contain the Escherichia coli tryptophan promoter-operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the “attenuator peptide” coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the “attenuator peptide” SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.     

Providing or hiding information: On the evolution of amplifiers and attenuators of perceived quality differences

In many coevolutionary systems members of one party select members of a second party based on quality differences existing among members of the latter (e.g., predators and prey, pollinators and flowers, etc.). We examined the fate of characters that increase (amplifiers) or decrease (attenuators) the perceived amplitude of differences in the quality upon which choice of the selecting party is based. We found that the evolution of such characters depends on (i) the relationship between the cost of the character and the relative benefit it gives to the high quality individuals (if an amplifier) or low quality individuals (if an attenuator), and (ii) the frequency, among members of the selected party, of the quality sought by the selecting party.     

Sequence and deletion analysis of the recombination enhancement gene (ref) of bacteriophage P1: evidence for promoter-operator and attenuator-antiterminator control.

The ref gene of bacteriophage P1 stimulates recombination between two defective lacZ genes in the Escherichia coli chromosome (lac x lac recombination) and certain other RecA-dependent recombination processes. We determined the DNA sequence of the 5' portion of the ref gene and tested various regions for functionality by inserting DNA fragments lacking increasing amounts of 5' sequence into plasmid and lambda phage vectors and measuring the ability of the constructs to stimulate lac x lac recombination. The region found essential for Ref activity in the absence of external heterologous promoters encodes two presumptive promoters, pref-1 and pref-2, whose -10 regions fall in a nearly perfect 13-base-pair (bp) tandem repeat. The -10 region of the putative pref-1 is part of a phage P1 c1 repressor recognition sequence. The first two ATG codons in the ref reading frame are, respectively, 90 and 216 bp downstream from the putative promoter-operator region. Deletion analysis indicated that translation can initiate at either ATG (although neither is associated with a canonical ribosome-binding sequence) and that the 42 amino acids in between are not indispensable for Ref stimulation of lac x lac recombination. However, the shorter reading frame appears to encode a less active polypeptide. The 91-bp leader region between the putative promoter-operator and the first ATG contains 30 codons in frame with the ref structural sequence, but its frame can be shifted without affecting Ref activity. The leader region ends with an apparent rho-independent termination sequence (attenuator). Deletion of 18 bp of early leader sequence drastically reduced Ref activity, even when ref was driven by a heterologous promoter (plac). An 8-bp internal deletion in the putative attenuator sequence relieved this requirement for the early leader sequence. This latter observation, along with nucleotide complementarity between portions of the early leader and attenuator sequences, are consistent with preemption of attenuation by the early leader.