Inhibition By a Downstream Element

Inhibition By a Downstream Element

A lambda phage not integrated into the host chromosome and proceed-ing down the lytic pathway would do well to minimize its synthesis of Int protein. On the other hand, an induced lambda located in the chromosome has great need for Int protein to assist in excision. How could the phage know whether or not it is integrated into the chromo-some and how could it therefore adjust Int protein synthesis? In both situations, the int-specific promoter p_I is off and the synthesis and regulation of Int and Xis proteins derives from the p_L promoter.

The location of the b2 region of the phage signals whether it is integrated in the chromosome or is free in the cytoplasm. When the phage has excised it is circular, and the b2 region is adjacent to the int gene (Fig. 18.12). On the other hand, when the phage is integrated, host DNA is adjacent to the int gene. These two situations have different

Figure 18.12 Sibis not part of theintmessenger when lambda is transcribedas a lysogen.

effects on the translation efficiency of int messenger deriving from the p_Lpromoter. A sequence called sib lying in the b2 region is transcribedand becomes part of the int messenger only when the phage is not integrated. This sequence inhibits translation of the int message.

The synthesis of Int and Xis proteins was characterized in cells that had been exposed with a low dose of UV light to block host-encoded protein synthesis. These cells were then infected with phage, and radio-active amino acids were added. At various times, samples were taken and subjected to SDS polyacrylamide gel electrophoresis. The Int and Xis proteins were easily detected and quantitated by autoradiography. Alternatively, no UV light was used, and instead the Int or Xis proteins were precipitated with antibody, further separated from the other proteins by the electrophoresis, and finally quantitated from autora-diographs.

Infection by wild-type lambda yields only a low synthesis of Int protein, but infection by λb2 yields high synthesis of Int protein. Point mutations and deletions have been isolated that yield high levels of Int protein following infection. These narrowly define a region in the b2 region called sib which is responsible for the altered synthesis rate of Int. The deletions and point mutations have a strictly cis effect on Int protein synthesis. Therefore the effect is called downstream or retro-in-hibition because it acts backward on expression of the preceding gene.

The sib region contains a typical transcription termination sequence, and the sib mutations alter the structure of the RNAse III-sensitive structure that can be formed in messenger that initiates from p_L (Fig. 18.13). Transcripts initiated at p_I normally terminate near the end of this region, but the transcripts initiated at p_L ignore termination signals because of the phage N protein bound to RNA polymerase, and the

Figure 18.13 Secondary structures of thesibregion in transcripts originatingfrom pI and pL. The promoter pI lies just ahead of xis, so the transcription start point does not include all of xis.

transcription continues past this region. The p_L-initiated transcripts are capable of forming an additional base-paired region that is sensitive to RNAse III. This enzyme that cleaves special double-stranded RNA structures. The transcripts initiated at p_I do not form the RNAse III-sen-sitive structure. Most likely, then, cleavage by RNAse III leads to degra-dation of the adjacent int gene messenger. As expected, the sib effect is absent in RNAse III-defective mutants.