Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements

An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5'-AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR, was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20-28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function.     

nutR中的突变对λ噬菌体早期转录抗终止作用的影响

将λＤＮＡ的ｎｕｔＲ序列置于Ｌａｃ启动子的下游,在ｎｕｔＲ和β－半乳糖苷酶基因（ｇａｌＫ）之间插入λ噬茵体依赖ｒｈｏ的终止子ｔＲ１,使ｇａｌＫ的表达取决于Ｎ蛋白介导的转录抗终止作用。为研究ｎｕｔＲ对抗终止的影响,系统地进行了该序列中每个碱基的点突变（Ａ→Ｃ,Ｇ→Ｔ,Ｃ→Ａ及Ｔ→Ｇ）．结果表明ｂｏｘＡ中有两个碱基（位置２和５）的突变对抗终止作用是至关重要的,使抗终止效率降低了１０倍;而其他位置的改变影响甚微。ｂｏｘＡ的缺失在ｎｕｓ￣＋宿主中使抗终止数率降低了４０％,但在ｎｕｓＢ宿主中却恢复到野生型水平。ｂｏｘＢ茎环结构中,茎部顶端的碱基对及环中邻近茎部的两个碱基的突变对抗终止作用影响很大,被认为是Ｎ蛋白的识别序列．ｂｏｘＡ和ｂｏｘＢ之间的间隔序列中有两个碱基的突变几乎使抗终止作用丧失。     

Interaction of the H4 autonomously replicating sequence core consensus sequence and its 3''-flanking domain.

Yeast autonomously replicating sequence (ARS) elements are composed of a conserved 11-base-pair (bp) core consensus sequence and a less well defined 3'-flanking region. We have investigated the relationship between the H4 ARS core consensus sequence and its 3'-flanking domain. The minimal sequences necessary and sufficient for function were determined by combining external 3' and 5' deletions to produce a nested set of ARS fragments. Sequences 5' of the core consensus were dispensable for function, but at least 66 bp of 3'-flanking domain DNA was required for full ARS function. The importance of the relative orientation of the core consensus element with respect to the 3'-flanking domain was tested by precisely inverting 14 bp of DNA including the core consensus sequence by oligonucleotide mutagenesis. This core inversion mutant was defective for all ARS function, showing that a fixed relative orientation of the core consensus and 3'-flanking domain is required for function. The 3'-flanking domain of the minimal functional H4 ARS fragment contains three sequences with a 9-of-11-bp match to the core consensus. The role of these near-match sequences was tested by directed mutagenesis. When all near-match sequences with an 8-of-11-bp match or better were simultaneously disrupted by point mutations, the resulting ARS construct retained full replication function. Therefore, multiple copies of a sequence closely related to the core consensus element are not required for H4 ARS function.     

Role of conserved sequence elements in yeast centromere DNA.

Conserved sequence features in Saccharomyces cerevisiae CEN DNA are confined to a region of approximately 120 bp. The highly conserved 8 bp at the left (PuTCACPuTG) constitute the left boundary of a functional CEN DNA as shown by the analysis of a series of Bal31 deletions. The right boundary of a functional CEN DNA lies within the conserved 25 bp at the right (TGT-T-TG--TTCCGAA-----AAA) or a few base pairs further outside of the 120-bp region. One mutant which just lacks the left conserved DNA element PuTCACPuTG can still assemble into a partially functional mitotic centromere and it assembles into a well functioning meiotic centromere. The sequences between the two conserved terminal DNA elements can be increased in length (+50%) or in GC content (from 6% to 12%) without measurable changes in mitotic and meiotic segregations of plasmids carrying such CEN mutations. The naturally occurring length and GC content of this centromere DNA sequence element is, therefore, not essential for centromere function. We discuss the possibility that it partly acts as a hinge region between two domains. Finally, we tested integrations of CEN DNA into the genome and found a toleration of wild-type CEN6 DNA when present 3' of the LYS2 gene.     

RNA-binding specificity of E. coli NusA

The RNA sequences boxA, boxB and boxC constitute the nut regions of phage λ. They nucleate the formation of a termination-resistant RNA polymerase complex on the λ chromosome. The complex includes E. coli proteins NusA, NusB, NusG and NusE, and the λ N protein. A complex that includes the Nus proteins and other factors forms at the rrn leader. Whereas RNA-binding by NusB and NusE has been described in quantitative terms, the interaction of NusA with these RNA sequences is less defined. Isotropic as well as anisotropic fluorescence equilibrium titrations show that NusA binds only the nut spacer sequence between boxA and boxB. Thus, nutR boxA5-spacer, nutR boxA16-spacer and nutR boxA69-spacer retain NusA binding, whereas a spacer mutation eliminates complex formation. The affinity of NusA for nutL is 50% higher than for nutR. In contrast, rrn boxA, which includes an additional U residue, binds NusA in the absence of spacer. The K_d values obtained for rrn boxA and rrn boxA-spacer are 19-fold and 8-fold lower, respectively, than those for nutR boxA-spacer. These differences may explain why λ requires an additional protein, λ N, to suppress termination. Knowledge of the different affinities now describes the assembly of the anti-termination complex in quantitative terms.     

Extent of the DNA sequence required in integration of staphylococcal bacteriophage L54a.

We characterized the minimum length of the DNA sequence of the attachment sites involved in the integrative recombination of staphylococcal bacteriophage L54a. A DNA fragment carrying the functional viral attachment site (attP) or the bacterial attachment site (attB) was sequentially trimmed, recloned, and tested for integrative recombination in vivo. The size of the functional attP site was at least 228 base pairs (bp) but no more than 235 bp. The left endpoint of the attP site was located to between positions -142 and -140, whereas the right endpoint was located to between positions +86 and +93 with respect to the center of the core sequence. The attB site was located to within a 27-bp sequence, from position -15 to +12, which included the 18-bp core sequence.