The CYCI-239-0 mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu-replacement of the normal -Ala-Gly-sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-l-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYCI+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYCI-239-0 mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYCI mRNA diminishes translation by inhibiting elongation.During the past 20 years, the CYCI gene and its gene product iso-l-cytochrome c in the yeast Saccharomyces cerevisiae have been the subject of numerous studies. Mutationally altered forms of at least partially functional iso-icytochromes c have been obtained from over 500 intragenic revertants of various cycl mutants. The vast majority of these altered iso-1-cytochromes c are found at a level corresponding to the level of normal iso-l-cytochrome c in wild-type strains. Thus, most changes of the amino acid sequence do not appreciably affect the level of the protein.One type of exception is the revertants that contain AUG initiator codons situated at certain abnormal positions (25, 26, 33; S. B. Baim, Ph.D. Thesis, University of Rochester, Rochester, N.Y., 1984; S. Baim and F. Sherman, unpublished data). In this investigation we describe another type of exception, the cycl-239 revertants which contain only 20o of the normal level of iso-1-cytochrome c and contain a sequence which can accommodate the formation of a hairpin structure within the translated region of the mRNA.MATERIALS AND METHODS Genetic analysis. The symbol CYCI+ or CYCI denotes the wild-type structural gene that encodes iso-l-cytochrome c. Mutations of this gene that cause a deficiency in either the amount or activity are designated by the symbol cycl followed by the allele number, e.g., cycl-17, cycl-239, etc. Intragenic revertants of specific cycl mutants in which the amount or activity of iso-l-cytochrome c has been at least partially restored are designated by the gene symbol CYCI followed by the allele number of the mutation from which they were derived and by capital letters to designate independent revertants. For example, CYCI-239-A, CYCI-239-B, etc., denote intragenic revertants of cycl-239.Conventional Zaret and Sherman (38,39). Briefly, logarithmic-phase cells from an overnight culture were inoculated at various densities into YPD (1% yeast extract [Difco], 2% Bacto-Peptone, and 2% glucose) medium. The cultures were shaken vigorously at 30°C for 18 to 24 hours. The cells were harvested at the appropriate densities and disrupted with glass beads. The lysate was adjusted to 1% sodium dodecyl sulfate and extracted several times with phenol-chloroform-isoamyl alcohol (25:24...

E2F is a cellular DNA-binding factor. Its binding activity is changed within adenovirus-infected cells so that it binds cooperatively to pairs of properly spaced and oriented E2F recognition sites. In the work described in this report, the conversion to cooperative binding was shown to require the adenovirus E4 17-kilodalton (kDa) polypeptide. Mutant viruses carrying alterations within the E4 17-kDa coding region failed to generate the infection-specific, cooperatively binding form of E2F. It was possible to alter E2F from uninfected cells so that it bound cooperatively by incubation with a partially purified fraction obtained from infected cells. The E4 17-kDa protein copurified with this activity and was also found to be present in a complex containing E2F. Consistent with its ability to alter the binding of E2F to its recognition sites within the E2 promoter, the E4 17-kDa polypeptide contributed to maximal expression of E2 mRNAs in some cell types. Its ability to enhance E2 transcription did not require expression of the ElA transactivator protein. These results are consistent with a model which proposes that the E4 17-kDa polypeptide binds to the cellular E2F factor, altering its binding behavior and thereby enhancing its ability to stimulate transcription. Three different early adenovirus gene products have been shown to activate the expression of other viral genes in trans. The best-studied activator is the ElA 289R protein. This ElA product is required for efficient activation of all remaining early viral genes (4, 28), and it functions to enhance the rate of transcription (36). The other activating products are the E1B 21-kilodalton (kDa) polypeptide (23, 51, 54), which has not been shown to act at the level of transcription, and an E4 gene product (13, 49). The mechanism of transcriptional activation by the ElA protein is not yet clear (for reviews, see references 3, 11, and 40). It has been proposed that ElA-mediated activation of the adenovirus E2 early promoter involves the induction or "activation" of a cellular factor termed E2F (37, 38, 40). E2F is a sequence-specific DNA-binding activity that recognizes the sequence 5'-TTTC(G/C)CGC-3' (30). There are 2345

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Under conditions in which cytoplasmic accumulation of HeLa cell mRNAs has been blocked by adenovirus infection, hsp70 family mRNAs are transported from the nucleus to the cytoplasm at near normal efficiency subsequent to heat shock. Heat shock does not reverse the general virus-induced block to host cell mRNA transport. The heat shock mRNAs are translated within the cytoplasm of the infected cell but at substantially reduced efficiency compared with that of uninfected cells. Thus, the hsp70 family of mRNAs can escape the transport block but not the translational block instituted late after adenovirus infection. The I-tubulin gene family is induced by the viral ElA gene after infection, and its mRNAs also accumulate in the cytoplasmic compartment. Given these two examples, it seems likely that the process of transcriptional induction allows the resulting mRNA to escape the viral block of transport.When procaryotic or eucaryotic cells are subjected to stress such as severely elevated temperature, they respond by rapidly synthesizing a small number of proteins, the so-called heat shock or stress proteins (reviewed in references 1, 9, 30, 39, and 44). One group of heat shock proteins related in amino acid sequence is termed the HSP70-like family. Cross-hybridization analysis suggests that this family contains at least 10 members, although these estimates include at least one pseudogene (17,36,47). DNAs coding four members of this family have been cloned from mammalian cells, and their products have been termed (39) HSP70, which is not detected in unstressed cells (32, 50); HSC70, which is constitutively synthesized and weakly induced upon stress (38, 47); GRP78, a constitutive but inducible component of the endoplasmic reticulum (28, 37); and HSX70, which is constitutively synthesized but further induced by stress (53), the adenovirus ElA protein (25, 54), or serum (55). We used the preceding designations in this report in the absence of an accepted uniform terminology for these genes.We used the heat-inducible HSP70 family as a test system to explore the fate of an induced cellular transcription unit late after adenovirus infection. Cellular gene expression is shut off at two levels within adenovirus-infected cells. Host cell mRNAs continue to be transcribed and processed in the nucleus but fail to appear in the cytoplasm (2, 6). This effect is mediated by a protein complex that includes two adenovirus-coded products, the ElB-55K and E4-34K polypeptides (4,16,31,41,42,51). The second level of shutoff occurs in the cytoplasm of the infected cell, where host cell mRNAs are displaced from active polysomes (e.g., see reference 40).When the hsp70 gene family is induced in adenovirusinfected HeLa cells, heat shock mRNAs accumulate within the cytoplasm at nearly the same efficiency as in uninfected cells. The hsp70 mRNAs are poorly translated, however, within the infected-cell cytoplasm. The mRNA from a sec-* Corresponding author.ond induced gene (P-tubulin transcription is induced by E1A; 48) also reaches the cytoplasm o...