TRANSLATIONAL REGULATION BY BACTERIOPHAGE X 
Gabriel Guarneros-Pena, Ph.D., International Research Scholar 
There are few documented cases of control of 
host-cell protein synthesis by bacteriophage. Dr. 
Guarneros and his colleagues have focused their re- 
search on translational control events in which A 
phage-directed transcripts act on protein synthesis 
at a step controlled by peptidyl-tRNA hydro- 
lase (Pth). 
Bacteriophage X is unable to grow vegetatively on 
bacterial mutants for the pth gene. Phage mutants 
that compensate the hydrolase defect defined sev- 
eral sites in the X genome. In an effort to understand 
this relationship, the phage bar mutation and the 
bacterial pth mutations have been studied. 
It has been known for some time that Pth is essen- 
tial for the cell. Bacteria carrying a thermosensitive 
mutation in pth accumulate peptidyl-tRNAs, stop 
protein synthesis, and die at the nonpermissive tem- 
perature. It is accepted that Pth scavenges peptidyl- 
tRNAs that fall oflf the ribosomes in translational 
editing. This view assumes that the accumulation of 
peptidyl-tRNAs is toxic for the cell. 
Characterization of the Bacterial 
and X Products Involved 
in the Regulatory Interaction 
Sequencing analysis in the laboratory of Dr. Guar- 
neros identified a translational open reading frame 
with the pth gene. Mutations that conferred the 
phage exclusion phenotype were 1-bp substitutions 
in pth. The Pth activity' was isolated and purified. 
The properties of the polypeptide corresponded to 
the predicted global amino acid composition, 
amino-terminal sequence, molecular weight, and 
isolelectric pH. In addition, in collaboration with 
Dr. Richard Buckingham in Paris, it was shown that 
the assigned polypeptide had Pth activity. 
The X mutations that overcome the Pth defect 
were located to several bar loci. The sequence anal- 
ysis of mutations in two discrete loci defined a 
nearly identical l6-bp DNA segment containing 
dyad symmetry. Genetic evidence suggested that the 
inhibition of X growth in mutant pth cells required 
transcription of the bar region. This supposition 
proved to be correct in a plasmid system. 
X Bar Transcription May Be Lethal 
to pth Bacteria 
Plasmid constructs containing a X bar sequence 
under an active promoter phenotypically parallel 
phage exclusion in Pth-defective cells. Transcrip- 
tion through wild-type X bar sites was lethal to Pth- 
defective (but not to wild-type) cells, whereas tran- 
scription of the mutant bar sites was harmless. This 
result does not necessarily imply that phage growth 
inhibition occurs by increased pth mutant lethality. 
Plasmid clones carrying bar sequences as small as 
2 1 bp and containing the 1 6-bp core caused lethal- 
ity of pth mutants. Most likely, transcripts are the 
active molecules, since there is no evidence of trans- 
latability of these sequences. Transcription of bar is 
followed by a general arrest of protein synthesis in 
the cell. Since RNA synthesis is not blocked, the inhi- 
bition must be a post-transcriptional effect. It is 
likely that inhibition of protein synthesis causes the 
lethality of Pth-defective cells. 
A Model for X Regulation of Protein 
Synthesis 
The Bar transcripts may stop protein synthesis by 
interfering with translation termination. Drs. Eman- 
uel Murgola, Albert Dahlberg, and their collabora- 
tors have implicated antiparallel pairing of 1 6S ribo- 
somal RNA and UGA termination codons in mRNA as 
part of the normal process of peptide synthesis ter- 
mination. Such base-pairing facilitates peptidyl- 
tRNA hydrolysis by preventing amino acid misincor- 
poration at UGA (suppression) . 
Drs. Murgola and Guarneros have proposed that 
Bar RNA competes with the UGA codons in mRNA 
for association with ribosomal 16S RNA. This would 
leave UGA codons in mRNA available for suppres- 
sion, therefore blocking efficient peptidyl-tRNA hy- 
drolysis. The proposal includes the participation of 
Pth in the translation termination step as a result of 
the association of the hydrolase with Bar regulation. 
Inspection shows that the transcripts of the core bar 
sequences contain a UGA codon within a five- 
nucleotide tract capable of antiparallel pairing with 
ribosomal 1 6S RNA. Mutant Pth may cause a defect 
in polypeptide termination by facilitating Bar RNA- 
16S RNA interaction. Results of non-sense suppres- 
sion in Pth-defective bacteria and of UGA-specific 
Bar-mediated suppression in wild-type cells support 
the basic predictions of the model. 
Role of Bar Regulation in X Biology 
The results discussed above for the bar plasmids 
in Pth-defective bacteria cannot be directly applied 
to explain the role of Bar RNA in X biology. How- 
ever, assuming either of the two functions advanced 
for Pth, scavenging of free peptidyl-tRNAs or transla- 
tion termination. Dr. Guarneros and his colleagues 
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