On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli.

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.

Influence of the relA gene on ribosome frameshifting.

We have examined the influence of genotype at the relA locus on the kinetics of leftward (or -1) frameshifting at a variety of codons calling for a limiting aminoacyl-tRNA species. We used lacZ left-frameshift reporter constructs carrying the sequenceU UUC XYZ, whereXYZ was each of three triplets coding for three different amino acids; we slowed the ribosomes at each of these by limiting for the amino acid or for the aminoacyl-tRNA. In all cases, limitation stimulated leftward frameshifting. In all cases, the stimulation was greater in relA mutant cells than in their wild-type relA(+) counterparts. In the latter genotype, the increased frameshifting was constant from the start of the limitation regime. This was also true of the relA mutant strain during limitation for lysine-tRNA or for leucine; however, during limitation for isoleucine-tRNA (or for isoleucine) the mutant showed a gradual, progressive increase in frameshifting, suggesting an indirect effect. We suggest that gradual accumulation of undermodified tRNAs, which is characteristic of the relA response, is involved. However, the specific modification involved is unknown. It is not queosine: analysis of a tgt mutant that is completely defective in queosine modification showed no increase in leftward frameshifting on the reporter which showed the larger, gradual increase during the relA response to isoleucine-tRNA limitation.

Ribosomes can slide over and beyond "hungry" codons, resuming protein chain elongation many nucleotides downstream.

In cells subjected to moderate aminoacyl-tRNA limitation, the peptidyl-tRNA-ribosome complex stalled at the "hungry" codon can slide well beyond it on the messenger RNA and resume translation further downstream. This behavior is proved by unequivocal amino acid sequence data, showing a protein that lacks the bypassed sequence encoded between the hungry codon and specific landing sites. The landing sites are codons cognate to the anticodon of the peptidyl-tRNA. The efficiency of this behavior can be as high as 10-20% but declines with the length of the slide. Interposition of "trap" sites (nonproductive landing sites) in the bypassed region reduces the frequency of successful slides, confirming that the ribosome-peptidyl-tRNA complex passes through the untranslated region of the message. This behavior appears to be quite general: it can occur at the two kinds of hungry codons tested, AUA and AAG; the sliding peptidyl-tRNA can be any of three species tested, phenylalanine, tyrosine, or leucine tRNA; the peptidyl component can be either of two very different peptide sequences; and translation can resume at any of the three codons tested.

Enhanced ribosome frameshifting in stationary phase cells.

We have examined the effect of growth phase in Escherichia coli on the translation of a plasmid-borne lacZ gene in which active enzyme synthesis requires a leftward frameshift. During the log phase of growth, the differential rate of enzyme synthesis is very low. It increases by about two orders of magnitude during the small amount of protein synthesis which occurs at the end of log phase and the early part of stationary phase. The increase is sufficient to increase the enzyme's specific activity in crude extracts to 30 times more than it would be if the log-phase differential rate continued unchanged. No such large increase is observed with a zero-frame lacZ+ control gene on the same plasmid under the control of the same promoter; a significant but much smaller increase is observed with a zero-frame control containing an in-frame terminator triplet in the region of the required frameshift. Protein sequence analysis of the enzyme made from the frameshift reporter in stationary cells shows that the increased enzyme synthesis is due to frameshifting, and not due to termination and reinitiation. The frameshift occurs at or right after the sequence U UUC AAG, an intrinsically shifty site.

On the mechanism of leftward frameshifting at several hungry codons.

We have used lacZ reporter genes to assess leftward ribosome frameshifting on sequences containing the quadruplet U UUC followed by several different triplets coding for lysine, isoleucine, or leucine. Limitation for lysine-tRNA provokes leftward frameshifting when the slippery quadruplet is followed by either lysine codon aag or aaa, but not when followed by an isoleucine or leucine codon. Limitation for isoleucine provokes frameshifting when the quadruplet is followed by either isoleucine codon aua or auc, but not when it is followed by a lysine codon. We conclude that the quadruplet promotes shifting when the ribosome is stalled at any "hungry" codon immediately after it. Changing the quadruplet to U AGC, at which peptidyl-tRNA cognate to the AGC triplet will be mismatched at all three anticodon positions if it slips left, abolishes frameshifting when the ribosome is stalled at the next position. We conclude that the U UUC quadruplet promotes frameshifting by virtue of its ability to pair with a left-slipped peptidyl-tRNA. The frameshift promoted by isoleucine-tRNA limitation of the U UUC aua sequence was analyzed by amino acid sequencing of the protein product. It occurs through reading of the Cau histidine codon overlapping the hungry codon from the left. This result rules out a "simultaneous slippage" type of mechanism. It strongly suggests instead that starvation-promoted frameshifting occurs primarily by slippage of peptidyl-tRNA just upstream of the stall site, followed by decoding of the triplet overlapping the stall site from the left or 5' side. A secondary finding is that the last base of the "hungry" codon has a moderate effect on its shiftiness, aag being shiftier than aaa, and aua being shiftier than auc.

The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli.

A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylalanine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closely related frameshift mechanisms. In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage' mechanism. In the second, ribosomes appear to slip before binding aminoacyl-tRNA, and then bind phenylalanyl-tRNA, which is encoded in the left-shifted reading frame. This mechanism is identical to the 'overlapping reading' we have demonstrated at other bacterial frameshift sites. The HIV-1 sequence is prone to frame-shifting by both mechanisms in E. coli.

On the directional specificity of ribosome frameshifting at a "hungry" codon.

Limitation for aminoacyl-tRNA promotes ribosome frameshifting at certain sites. We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context. Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left. Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rightward frameshifting, demonstrated here, permits us to define a minimal heptanucleotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply.

On the role of the P-site in leftward ribosome frameshifting at a hungry codon.

Previous work characterized ribosomal frameshifting within the sequence C UUC AAG provoked by lysyl-tRNA limitation. The ribosome frameshift is one base to the left of the AAG lysine codon, as shown by dotted overlining above. We now show that the frequency of this leftward ribosome frameshift is strongly influenced by the identity of the bases two, three and four positions to the left of the actual frameshift site. The nature of these influences coincides exactly with the possibilities of base-pairing between the sequence and the anticodon of the P-site peptidyl-tRNA when shifted one base to the left just upstream of the frameshift site. We conclude that a peptidyl shift in the P-site is intimately involved in leftward frameshifting in the adjacent A site when it codes for an aminoacyl-tRNA in short supply.

Context rules of rightward overlapping reading.

We have investigated the mechanism and sequence context rules governing ribosome frameshifting promoted by aminoacyl-tRNA limitation. In the case of one shifty sequence, frameshifting promoted by lysyl-tRNA limitation occurs at the sequence AAG C and is due to rightward movement of the ribosome so as to read the AGC triplet overlapping the hungry codon from the right. The frequency of this event is unaffected by sequence elements more than three bases to the left (upstream) or two bases to the right (downstream) of the hungry codon, and only slightly affected by the identity of the base two bases to the right. It is strongly affected by the base immediately to the right of the hungry codon, which becomes the wobble base of the shifted triplet; and by the third base of the hungry codon, even though the two synonyms (AAG and AAA) call for the same aminoacyl-tRNA; and by the identity of the base immediately to the left of the hungry codon. The latter result suggests that the aminoacyl-tRNA in the P site affects the maintenance of reading frame at the adjacent A site of the ribosome. However, the DNA sequence makes it seem unlikely that the P-site tRNA shifts to the right in concert with the A-site tRNA, a mechanism that can account for leftward frameshifting (in the opposite direction) in retroviral translation. The specificity of sequence determinants of leftwing versus rightwing frameshifting is discussed.

Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.

A prospective study of aspirin's effect on red blood cell loss in cardiac surgery.

The effect of aspirin on red blood cell (RBC) loss and blood transfusions was evaluated prospectively in 100 consecutive patients, with normal bleeding times, undergoing elective coronary artery bypass (CABG) surgery. Patients taking 85-325 mgm of aspirin daily up to or within 48 hours of surgery (the "aspirin" group) were compared to patients not taking aspirin or those who had discontinued aspirin at least 4 days before surgery (the "no-aspirin" group). RBC loss was determined by measuring preoperative and postoperative RBC volume using RISA and 51Cr techniques. There were no significant differences, respectively, between the aspirin and no-aspirin groups for: RBC loss (1158 +/- 67 ml vs 1129 +/- 47 ml, p = 0.737), chest tube drainage (925 +/- 31 ml vs 844 +/- 70 ml, p = 0.553), and gm% discharge Hemoglobin (Hgb) (9.94 +/- 0.32 vs 9.49 +/- 1.4, p = 0.0148). Strict criteria for blood transfusions were employed: (1) intraoperative hematocrit of less than 21%, (2) postoperative Hgb of less than 7 gm% for patients less than 70 years old and (3) postoperative Hgb of less than 8 gm% for patients greater than 70 years old. There were no significant differences, respectively, between the aspirin and no-aspirin groups for units of blood transfused (1.32 +/- vs 1.21 +/- 0.20, p = 0.843) and patients not receiving transfusions during the entire hospitalization (44% vs 50%). Patients taking 85-325 mgm of aspirin with a normal bleeding time undergoing elective CABG did not have increased RBC loss or increased transfusion requirements. These results indicate it is not necessary to delay elective CABG surgery for the purpose of discontinuing aspirin.

Effects of intraoperative plasmapheresis on blood loss in cardiac surgery.

Intraoperative platelet-rich plasmapheresis allows autotransfusion of fresh, undamaged platelets and clotting factors at the completion of the operation. To evaluate this technology, we randomly assigned 100 consecutive patients who were to undergo an elective coronary bypass procedure and had normal clotting studies into the experimental (plasmapheresis) or the control group. Characteristics of both groups were similar, including average age (61.4 years versus 61.3 years [experimental versus control group]), sex (78% male versus 74% male), preoperative weight (80.9 kg versus 80.2 kg), preoperative red cell mass (1,989 mL versus 1,890 mL), perfusion time (102 minutes versus 106 minutes), and coagulation studies. Both internal mammary arteries were used in 68% of the patients. All patients had preoperative and postoperative blood volume determinations and complete clotting studies. Sixty-two variables related to bleeding were analyzed. Strict indications for transfusion were a hemoglobin level less than 7 g/100 mL in patients younger than 70 years and a hemoglobin level less than 8 g/100 mL in patients older than 70 years. The group receiving intraoperative plasmapheresis had a significant reduction in operative red cell mass loss (1,050 +/- 43 mL versus 1,226 +/- 61 mL; p = 0.021), a reduction in the average homologous transfusion (0.67 +/- 0.15 unit versus 1.8 +/- 0.25 units; p = 0.0002), and an increase in the percentage of patients not requiring blood transfusions (66% versus 32%; p = 0.001). This technique is useful in reducing postoperative blood loss and homologous transfusions.

On the mechanism of ribosomal frameshifting at hungry codons.

In a few, rather rare cases, frameshift mutant alleles are phenotypically suppressed during limitation for particular aminoacyl-tRNA species. The simplest interpretation is compensatory ribosome frameshifting at a "hungry" codon in the vicinity of the suppressed frameshift mutation. We have now tested this interpretation directly by obtaining amino acid sequence data on such a phenotypically suppressed protein. We used a plasmid-borne lacZ gene, engineered to be in the (+) reading frame. Its background leakiness is increased by two orders of magnitude during lysyl-tRNA limitation. The enzyme made under this condition has the amino acid sequence expected from the DNA sequence up to the first lysine codon, then shifts in the (-) direction to recreate the correct lacZ reading frame. The lysine is replaced by serine, presumably due to cognate reading of an overlapping AGC codon displaced by one base to the 3' side of the AAG codon. When the 3' overlapping codon is AGA or AGG, there is no ribosome frameshifting; when it is AGU (read by the same serine tRNA) there is frameshifting, although less efficiently than in the case of AGC. The mechanism of cognate overlapping reading contradicts more elaborate models that two of the authors have suggested previously. However, the possibility remains that there is more than one mechanism of ribosome frameshifting at hungry codons.

Inseparability of X-Heterochromatic Functions Responsible for X:Y Pairing, Meiotic Drive, and Male Fertility in Drosophila melanogaster.

Deficiencies encompassing part or all of the X heterochromatin of Drosophila melanogaster have been linked to three abnormalities in male meiosis and spermatogenesis: X-Y nondisjunction, skewed sperm recovery ratios favoring sperm with reduced chromatin content, and sterility in males carrying either a Y-autosome translocation or mal( +)Y. In this study, 18 X heterochromatic deficiencies of varying sizes were tested in XY males for their spermatogenic phenotypes. All 18 proved to be either mutant for all three phenotypes or wild type for all three. Although variable among mutant deficiencies, expression levels of all three phenotypes were strongly correlated. Deficiencies that cause high levels of nondisjunction also cause severe recovery ratio distortion and are completely sterile in conjunction with mal(+) Y. Low nondisjunction deficiencies cause comparable mild effects for the other phenotypes. The same deficiencies were also tested in males carrying a large heterochromatic free X duplication Dp(1; f)3. For all deficiencies which induce nondisjunction in XY males, the Y and free duplication pair regularly and the X fails to pair in XYDp males. Drive levels are constant across deficiencies in these males. Thus elimination of variability in the pairing phenotype also eliminates variability in sperm recovery ratios.