Control of Bacterial Protein Synthesis 
During Viral Infection 
Gabriel Guameros Pena, Ph.D. — International Research Scholar 
Dr. Guameros is Professor of Genetics and Molecular Biology at the Center for Research and Advanced 
Studies, National Polytechnic Institute, Mexico City. He received his undergraduate and M.Sc. degrees in 
microbiology, chemistry, and biochemistry in Mexico City, and his Ph.D. degree in molecular biology 
from the University of California, Berkeley, where he studied with Harrison Echols. He joined the staff of 
the Center after doing postdoctoral work in molecular biology at the University of Geneva, Switzerland, 
in Harvey Eisen's laboratory. He has been awarded fellowships from the Guggenheim Memorial 
Foundation, the Commission of the European Communities, and the Sistema Nacional de 
Investigadores, Mexico. 
INFECTING viruses divert the functions of the 
host cell for their own development. To 
achieve this, the viral genome directs the synthe- 
sis of regulatory molecules, which reorient cell 
functions to conform to the specific viral develop- 
ment program. There is abundant evidence of 
transcriptional control during phage X infection 
of Escherichia coli, where the phage genome di- 
rects the synthesis of regulatory proteins that 
alter the cells' transcriptional pattern. Little is 
known, however, about the translational control 
in X-infected cells. Our laboratory has pursued a 
case of translational control involving X RNA se- 
quences, named bar, and peptidyl-tRNA hydro- 
lase (Pth), a bacterial enzyme essential for pro- 
tein synthesis. 
The Target in the Cell 
Bacterial mutants partially defective in the ac- 
tivity of Pth are unable to support the growth of X 
phage. We have shown that the corresponding 
mutations are located in the pth gene region. This 
result was further confirmed by sequence analy- 
sis; the mutations are base substitutions within a 
translational open reading frame that corre- 
sponds to the pth gene. Pth, the gene product, 
was isolated and characterized chemically and 
enzymatically. 
Pth hydrolyzes peptidyl-tRNAs to yield free 
tRNAs and peptides. It has been proposed that the 
enzyme is a scavenger of peptidyl-tRNAs that have 
dropped off the ribosomes during editing of mis- 
incorporated amino acids in polypeptide chains. 
The Pth function is essential for the cell, as in- 
ferred from the fact that a heat-sensitive mutant of 
Pth accumulates peptidyl-tRNAs and stops pro- 
tein synthesis upon shift to the nonpermissive 
temperature. Moreover, the enzyme is ubiquitous 
among organisms from bacteria to mammals. 
The possible role of Pth in ribosome-bound hy- 
drolysis of peptidyl-tRNA at the step of polypep- 
tide chain termination has not been supported by 
the work of others. Preliminary results, however, 
indicate that Pth may be involved in polypeptide 
chain termination (see below), and we aim to 
investigate this possible participation. 
Nature of the X Regulator 
We have isolated phage mutants that overcome 
the bacterial Pth defect. These mutations defined 
several genetic sites named bar. DNA sequence 
analysis of two of these, barl and barll, revealed 
that the mutations affect nearly identical l6-bp 
segments having dyad symmetry. 
The inhibition of phage development by mu- 
tants defective in Pth requires transcription 
through wild-type X bar regions. Transcripts 
themselves, not polypeptides, seem to be the ac- 
tive molecules. Aplasmid system in which short X 
bar sequences were cloned in front of an active 
promoter somehow mimics the X inhibition 
effect. 
Transcription of wild-type bar in plasmids is 
lethal to Pth-defective (but not wild-type) cells. 
This effect is specific, because constructs carry- 
ing mutant bar sequences are not lethal. Tran- 
scription through a vector harboring a synthetic 
nucleotide sequence as short as 20 bp mimicking 
barl caused pth mutant lethality; therefore we 
think that the 1 6-bp bar sequence is the core of 
the inhibitory transcripts. Protein synthesis is 
shut off soon after bar transcript induction, but 
RNA synthesis continues for several hours. Thus 
the lethal effect is probably caused by a rapid 
inhibition of protein synthesis. 
How does bar RNA inhibit protein synthesis? 
Preliminary data on nonsense codon-specific 
suppression, obtained in collaboration with 
Emanuel Murgola (M. D. Anderson Cancer Cen- 
ter, Houston), have led us to propose a unifying 
model that implicates foar RNAand Pth in peptide 
chain termination. The model postulates, first, 
that bar RNA can interfere with the termination 
of UGA-mediated translation by antiparallel base- 
pairing with ribosomal 1 6S RNA, and second, that 
mutant Pth causes a defect in polypeptide termi- 
nation facilitating bar RNA- 1 6S RNA interaction. 
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