A 5'-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

Abstract

The bacteriophage λ's cI mRNA was utilized to examine the importance of the 5'-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 5'-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 5'-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 5'-phosphate or a 5'-hydroxyl indicate that leaderless cI mRNA requires a 5'-phosphate for stable ribosome binding in vitro as well as expression in vivo. Ribosome-binding assays show that 30S subunits and 70S ribosomes do not bind as strongly to 5'-hydroxyl as they do to 5'-phosphate containing leaderless mRNA and the tRNA-dependent ternary complex is less stable. Additionally, filter-binding assays revealed that the 70S ternary complex formed with a leaderless mRNA containing a 5'-hydroxyl has a dissociation rate (k(off)) that is 4.5-fold higher compared with the complex formed with a 5'-phosphate leaderless mRNA. Fusion to a lacZ reporter gene revealed that leaderless cI mRNA expression with a 5'-hydroxyl was >100-fold lower than the equivalent mRNA with a 5'-phosphate. These data indicate that a 5'-phosphate is an important feature of leaderless mRNA for stable ribosome binding and expression.

FIGURE 1.

Hammerhead ribozyme sequence, proposed structure, and site of cleavage (arrow). Shaded regions represent areas of base pairing. (A) Structure resulting in leaderless cI-lacZ mRNA with a 5′-OH after cleavage. (B) Structure resulting in lac-leadered cI-lacZ mRNA with a 5′-OH after cleavage. Sequence in bold identifies the beginning of the cI coding sequence (A) or the lac-untranslated leader sequence (B).

FIGURE 2.

Mapping of mRNA 5′-ends by primer extension analysis of leadered (lanes 1,2) and leaderless (lanes 3,4) cI-lacZ fusions with a 5′-terminal hydroxyl or triphosphate. The closed arrows identify the 5′-end of the lac-untranslated leader (lanes 1,2) or the 5′-end (i.e., the translational start site) of LL mRNAs (lanes 3,4). The open arrows identify the expected migratory positions of uncleaved, hammerhead-containing mRNA. Lanes 2 and 3 show in vivo mRNA containing a 5′-hydroxyl, and lanes 1 and 4 show in vivo mRNA containing a 5′-triphosphate. Lanes labeled G, A, T, and C indicate the dideoxy termination sequencing reactions used to map the 5′ ends of transcripts.

FIGURE 3.

Analysis of cI-lacZ mRNA by Northern hybridization. Autoradiogram of size-fractionated in vivo RNA probed for cI-lacZ mRNA. Arrows indicate predicted positions of full-length mRNA (FL), 23S, and 16S rRNA. Lanes are labeled according to total in vivo RNA extracted from the E. coli RFS859 host strain (RFS) or the host strain containing plasmids expressing cI-lacZ fusions described in Table 1.

FIGURE 4.

Primer extension inhibition (toeprint) analysis of 30S subunit binding to SDL cI-lacZ mRNA with a 5′-triphosphate (lanes 1–5), a 5′-monophosphate (lanes 6–10), or a 5′-hydroxyl (lanes 11–15). Lanes 1, 6, and 11 omit 30S subunits, and lanes 2, 7, and 12 omit tRNA^fMet (1 μM when included). (Lanes 3,8,13) 30S subunit concentration (88 nM) is twofold higher than mRNA (44 nM) concentration. (Lanes 4,9,14) 30S subunit concentration (264 nM) is sixfold higher than mRNA concentration. (Lanes 2,5,7,10,12,15) 30S subunit concentration (528 nM) is 12-fold higher than mRNA concentration. The position of the toeprint signal is identified by an arrow.

FIGURE 5.

Primer extension inhibition (toeprint) analysis of 30S subunit binding to LL cI-lacZ mRNA with a 5′-triphosphate (lanes 1–6), a 5′-monophosphate (lanes 7–12), or a 5′-hydroxyl (lanes 13–18). Lanes 1, 7, and 13 exclude 30S subunits, and lanes 2, 8, and 14 exclude tRNA^fMet (1 μM when included). (Lanes 3,9,15) 30S subunit concentration (88 nM) is twofold higher than mRNA (44 nM) concentration. (Lanes 4,10,16) 30S subunit concentration (264 nM) is sixfold higher than mRNA concentration. (Lanes 5,11,17) 30S subunit concentration (528 nM) is 12-fold higher than RNA concentration. (Lanes 2,6,8,12,14,18) 30S subunit concentration (792 nM) is 18-fold higher than mRNA concentration. The position of the toeprint signal is identified by an arrow.

FIGURE 6.

Primer extension inhibition (toeprint) analysis of 70S ribosome binding to LL cI-lacZ mRNA with a 5′-triphosphate (lanes 1–4), a 5′-monophosphate (lanes 5–8), or a 5′-hydroxyl (lanes 9–12). Lanes 1, 5, and 9 exclude 70S ribosomes, and lanes 2, 6, and 10 exclude tRNA^fMet (1 μM when included). (Lanes 3,7,11) 30S subunit concentration (88 nM) is twofold higher than mRNA (44 nM) concentration. (Lanes 2,4,6,8,10,12) 70S ribosome concentration (264 nM) is sixfold higher than mRNA concentration. The position of the toeprint signal is identified by an arrow.

FIGURE 7.

Competition primer extension inhibition (toeprint) analysis of LL cI-lacZ mRNA bound to 70S ribosomes with or without tRNA^fMet. Prebound ternary complexes containing LL cI-lacZ mRNA with a 5′-monophosphate (A) or 5′-hydroxyl (B) are challenged with excess competitor mRNA containing a 5′-hydroxyl or a 5′-monophosphate. (A) Stability of ternary complexes containing 5′-phosphate LL mRNA. Lane 1 excludes ribosomes, and lane 2 excludes tRNA^fMet. (Lanes 3–5) The ternary complex incubated for 0, 20, and 30 min after the initial binding, respectively, with no addition of competitor mRNA. (Lanes 6–10) A 5′-hydroxyl LL mRNA competitor (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′phosphate mRNA (1X). (Lanes 11–15) A 5′-phosphate LL competitor mRNA (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′phosphate mRNA (1X). Reverse transcription was initiated at 0, 5, 10, 20, or 30 min after the competitor addition. (B) Stability of ternary complexes containing 5′-hydroxyl LL mRNA. Lane 1 excludes ribosomes, and lane 2 excludes tRNA^fMet. (Lanes 3,4) The ternary complex incubated for 0 and 20 min, respectively, after the complex formation, with no addition of competitor mRNA. (Lanes 5–8) A 5′-hydroxyl LL mRNA competitor (20X) was added to the reactions; (lanes 9–12) a 5′-phosphate LL mRNA competitor (20X) was added 15 min after initial ternary complex formation with primer-annealed 5′hydroxyl mRNA (1X). Reverse transcription was initiated at 0, 5, 10, and 20 min after the competitor addition. The position of the toeprint signals are identified by arrows.

FIGURE 8.

Filter binding analysis of tRNA^fMet dissociation from 70S ternary complexes with 5′-phosphate or 5′-hydroxyl LL cI-lacZ mRNA. Ternary complexes are formed with mRNA (0.5 μM), 30S subunits or 70S ribosomes (0.7 μM), and ³²P-labeled tRNA^fMet (≤0.05 μM). Reactions were diluted 100-fold into buffer containing 400-fold excess unlabeled tRNA^fMet. Samples were spotted onto a double membrane system over time (bottom). The nitrocellulose upper membrane shows tRNA^fMet bound to ribosomes (i.e., tRNA in ternary complex) and the positively charged lower membrane shows tRNA^fMet not in ternary complex (i.e., free tRNA). The graph shows the proportion of tRNA^fMet bound to ribosomes after phosphorimaging quantification of radiolabeled tRNA bound to each membrane and is used to derive the complex stabilities for radiolabeled tRNA in ternary complexes containing LL cI-lacZ mRNA with a 5′-phosphate (LL 5′P) or a 5′-hydroxyl (LL 5′OH), or ternary complexes containing SDL cI-lacZ mRNA with a 5′-phosphate (SDL 5′P).