Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

Abstract

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10-15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context.

Figure 1

NMR structures of RegB, RelE and YoeB endoribonucleases. The structures of RegB [29], RelE [144] and YoeB [145] are shown. The first α-helix of RegB, absent in the two other endoncleases, is drawn in pale orange. The two conserved α-helices are in red and orange and the conserved four-stranded β-sheet is in cyan.

Figure 2

Crystal structure of T4 RegA. In panel A, the RegA dimer (pymol rendering of PDB 1REG; [66]) is labeled at relevant structures discussed in the text. Panel B highlights the likely RNA binding residues in α helix 1 (K14, T18, R21) and loop residue W81. Also shown is the F106 residue that cross-links to bound RNA and is adjacent to the RNA binding region. See Figure 3 for the relative conservation of the labeled amino acids in other RegA proteins. Adapted from the data of [66,70,72].

Figure 3

Aligned RegA proteins of 26 T4-related phages. regA is immediately distal to gene 62 in the core DNA replication gene cluster of all T4-related genomes sequenced to date. Identity relative to T4 RegA is in column 2, aligned amino acids are shown using ClustalW colors, and dashes are gaps in the alignment. Residues numbered above the sequences reference the T4 protein. Asterisks mark the amino acids cited in the text as involved in RNA binding. At the bottom of the alignment are underlined structural elements of the protein from PDB 1REG[66]. Sequences were obtained from GenBank or the T4-like phage genome browser (http://phage.ggc.edu/).

Figure 4

Gene 32 translational repression site. In Panel A the leader mRNA for autogenous gp32 binding is shown for RB69, T4 and T2. The important TIR nucleotides are underscored with asterisks, the base-paired regions of the 5' pseudoknot are marked with arrows, and the T4 and RB69 regions bound by gp32 in protection assays are overlined [78]. Short nucleotide insertions in RB69 or T2 relative to T4 are in blue. Dashes (gaps) are inserted for alignment. Panel B is a cartoon-ribbon diagram of the T2 gene 32 pseudoknot diagramed in panel A that was obtained by multidimensional NMR methods [77]. Two A-form coaxially stacked stems are apparent. 5' and 3' terminal nucleotides are labeled. Jmol rendering used database entry 2 tpk. Figure was derived and adapted primarily from data in [77,78].

Figure 5

RNA structures affecting translation at T4-related phage TIRs. Panel A shows stem-loop regions that inhibit translation from early transcripts containing gene e. a) Phages are grouped if they have identical TIR regions. Other phages with e leaders identical to T4 include: T4T, T2, T6, RB18, RB26, RB32 and RB51. Those having gene e but no apparent RNA structure in the TIR: Aeh1, 44RR, 25, 31 & FelixO1. Phages examined but with no apparent gene e: RB16, RB43, RB49, phi-1, syn9, S-PM2, PSSM2, PSSM4, 65, 133, KVP40, nt-1, acj009, acj61. b) Gene e TIR nucleotides are marked with asterisks. Arrows mark the stems of the likely structures, which was demonstrated for T4 [98]. T4 bases noted with + are the mapped 5' transcript ends from the upstream late promoter (TATAAATA; shaded). Sequences were obtained from GenBank or the T4-type phage browser at http://phage.ggc.edu/. c) RNA folding and ΔG values were by the method of M. Zuker (http://mfold.rna.albany.edu/). Panel B shows the conserved stem at the gene 25 TIR of approximately 30 T-even related phages. Nucleotides of the TIR are indicated with an asterisk, with less conserved adjacent nucleotides noted with N. Panel B was derived from the data of [109].

Figure 6

PrrC tRNA anticodon nuclease and T4 exclusion system. A) The E. coli hsd gene cluster includes prrC. B) PrrC has N-terminal two thirds NTPase and EcoprrI interaction domains and, starting at residue 265, C-terminal tRNA recognition and ribonuclease (ACNase) catalytic domains. C) Current model for tRNA cleavage and T4 exclusion. i) PrrC, minimally as a head-to-tail dimer (tetramer and hexamer oligomers are possible) associates with EcoprrI on DNA, as an inactive, latent endoribonuclease. ii) In one of two allosteric activation mechanisms, EcoprrI-PrrC-DNA complex binds increased levels of dTTP and with GTP hydrolysis activated ACNase cleaves the anticodon of tRNA^Lys. iii) During infection, the small, T4-encoded polypeptide stp binds EcoprrI activating tRNA^Lys ACNase. iv) T4 repairs the cleaved tRNA at the 2',3'cyclic phosphate and 5' OH using polynucleotide kinase (Pnk) and RNA ligase (Rnl1). Figure adapted from the publications of Kaufmann and colleagues [111,114,116,119].

Figure 7

Model for programmed translational bypassing in T4 gene 60. A) In most T4-related phages, the large topoisomerase subunit (gp39) is encoded by a single gene. In T4, active site domains are interrupted by the mobA-60.1 coding region where the downstream ORF (gp60) contains the 50 nucleotide bypassed sequence. Elements in the mRNA that code for translational bypassing are shown. B) Recapitulation of the model [127] where E-, P- and A- sites of the ribosome are shown as translation enters the bypass region (blue mRNA). Nascent peptide is shown in gold and as promoting "take-off" from the green GGA codon. In (C) the bypass mRNA stem-loop structure occupies the A-site, precluding release factor 1 [66] from binding and inhibiting termination. Ribosomal protein L9 facilitates tRNA exit through the E-site and nascent peptide interactions prevent peptidyl-tRNA scanning. D) Landing site codon:anticodon pairing is shown, with entry of the resuming charged tRNA into the A-site for translating the distal region of gene 60 mRNA. The GAG six nucleotides 5' of the landing site is shown pairing with the 16S rRNA anti-SD region to help reinitiate peptidyl-tRNA scanning and pairing at the GGA. Figure adapted from [127], with kind permission of J. F. Atkins.