Translational repression by a transcriptional elongation factor

Abstract

One of the classical positive regulators of gene expression is bacteriophage lambda N protein. N regulates the transcription of early phage genes by participating in the formation of a highly processive, terminator-resistant transcription complex and thereby stimulates the expression of genes lying downstream of transcriptional terminators. Also included in this antiterminating transcription complex are an RNA site (NUT) and host proteins (Nus). Here we demonstrate that N has an additional, hitherto unknown regulatory role, as a repressor of the translation of its own gene. N-dependent repression does not occur when NUT is deleted, demonstrating that N-mediated antitermination and translational repression both require the same cis-acting site in the RNA. In addition, we have identified one nut and several host mutations that eliminate antitermination and not translational repression, suggesting the independence of these two N-mediated mechanisms. Finally, the position of nutL with respect to the gene whose expression is repressed is important.

Figure 1

Partial genetic map of bacteriophage λ showing genes (hatched boxes), cis-acting nut sites and DNA specifying the RNase III-sensitive hairpin (R-III) (▪), promoters (p_L and p_R), and transcriptional terminators (tL1 and tR1). Arrows represent transcriptional patterns in the absence and presence of N.

Figure 2

Structure of the N leader as predicted by Steege et al. (1987) showing NUTL, the RNase III-sensitive hairpin and cleavage sites, and the N Shine–Dalgarno sequence (SD) and initiation codon (N). Nucleotides are numbered from +1 of the p_L transcript.

Figure 4

N-mediated repression with different p_L–nutL–lacZ fusions. Each fusion contains the p_L promoter, the nutL site, and DNA sequence specifying the RNase III-sensitive hairpin (R-III) upstream of lacZ with different intervening sequence. (N′) The first 33 codons of the N structural gene; (sp) A spacer sequence containing translational stop codons. Numbers indicate the number of nucleotides between NUTL and the relevant Shine–Dalgarno sequence (SD) in the RNA. N⁻ cells carry pUC9 and N⁺ cells carry pNAS150 (p_lacN⁺). Shown are β-galactosidase activities in samples after 60 min of heat induction with zero time values subtracted. Data shown are averages of at least three experiments. The variability between averaged values is <21%.

Figure 3

The effect of N on the expression of a p_L–nutL–N–lacZ gene fusion. The fusion was present in single copy in the chromosome as part of a defective λ prophage. This strain carries either pUC9 (•) or pNAS150 (p_lacN⁺, ○). Shown are β-galactosidase activities in samples after indicated times of heat induction with the zero time value subtracted. Data shown are averages of at least two experiments. The variability between averaged values is <19%.

Figure 5

Analysis of N–lacZ RNA using RT–PCR. Primers were used to amplify a 1086-bp fragment of lacZ and 773 bp fragment of bioA on total RNA isolated from the p_L–nutL–N–lacZ gene fusion strain carrying pZH124(N⁺), pGB2(N⁻), or pZH126(Nun⁺). Samples are analyzed by electrophoresis on a 1.2% agarose gel. Numbers indicate the length in base pairs (×1000) of DNA markers. The level of N–lacZ mRNA in these samples can be normalized to the level of bio mRNA, which did not vary between N⁻ and N⁺ cells.

Figure 6

p_L–nutL–N–lacZ–galK double-reporter construct in which the expression of the gal operon is under the control of the λ p_L promoter. The expression of gal under N⁻ conditions is prevented by transcriptional terminators (T), including one in an IS2 element inserted in the gal leader sequence. N-mediated antitermination permits gal operon expression under N⁺ conditions. The gal operon is brought closer to p_L by the deletion pglΔ8, thus maximizing gal expression from p_L. The left-hand attachment site of the prophage is represented by att.

Figure 7

The nucleotide sequence of λ nut sites and relevant nutL mutations. nutLΔ eliminates the sequence shown plus 8 and 6 bp 5′ and 3′, respectively.

Figure 8

General models for N-mediated translational repression. (A) N is shown interacting with the NUTL–Nus factor complex associated with RNA polymerase as it passes the N gene. In this model the structure or action of the antitermination complex inhibits translation of N at the 5′ end of the p_L transcript. (B) N is shown interacting with NUTL to cause translational repression of N subsequent to the modification of RNA polymerase and release of NUTL from the antitermination complex.