Philip J Farabaugh

University of Maryland, Baltimore County | UMBC · Department of Biological Sciences

Protein-protein and protein–rRNA interactions at the interface between ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selection involves a major conformational change—domain closure—that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This ha...

Protein synthesis on the ribosome involves the successive rapid recruitment of cognate aminoacyl-tRNAs and rejection the much more numerous incorrect near or non-cognates. The principal feature of translation elongation is that at every step many incorrect aa-tRNAs unsuccessfully enter the A site for each cognate accepted. Normal levels of translat...

Saccharomyces cerevisiae has been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both appr...

Protein synthesis requires both high speed and accuracy to ensure a healthy cellular environment. Estimates of errors during protein synthesis in Saccharomyces cerevisiae have varied from 10-3 to 10-4 errors per codon. Here, we show that errors made by ${\rm{tRNA}}^{\rm Glu}_{\rm UUC}$ in yeast can vary 100-fold, from 10-6 to 10-4 errors per codon....

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The process of protein synthesis, termed translation, is complex, involving a large molecular machine, called the ribosome, that orchestrates the interaction of a large number of trans-acting factors, both RNA and protein. A large proportion of genes are translationally regulated with most of the regulation occurring at initiation of protein synthe...

Cellular health and growth requires protein synthesis to be both efficient to ensure sufficient production, and accurate to avoid producing defective or unstable proteins. The background of misreading error frequency by individual tRNAs is as low as 2 × 10−6 per codon but is codon-specific with some error frequencies above 10−3 per codon. Here we t...

Background A large number of Saccharomyces cerevisiae cellular factors modulate the movement of the retrovirus-like transposon Ty1. Surprisingly, a significant number of chromosomal genes required for Ty1 transposition encode components of the translational machinery, including ribosomal proteins, ribosomal biogenesis factors, protein trafficking p...

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caus...

##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##

The translation of genes into functional proteins involves error. Mistranslation is a known cause of disease, but, surprisingly, recent studies suggest that certain organisms from all domains of life have evolved diverse pathways that increase their tolerance of translational error. Although the reason for these high error rates are not yet clear,...

Protein synthesis must rapidly and repeatedly discriminate between a single correct and many incorrect aminoacyl-tRNAs. We have attempted to measure the frequencies of all possible missense errors by tRNAUUC(Glu), tRNAQUC(Asp) and tRNAQUA(Tyr). The most frequent errors involve three types of mismatched nucleotide pairs, U•U, U•C, or U•G, all of whi...

tRNA isopentenyltransferases (Tit1) modify tRNA position 37, adjacent to the anticodon, to N6-isopentenyladenosine (i6A37) in all cells, yet the tRNA subsets selected for modification vary among species, and their relevance to phenotypes is unknown. We examined the function of i6A37 in Schizosaccharomyces pombe tit1+ and tit1-Δ cells by using a β-g...

Ty3 elements of S. cerevisiae contain two overlapping coding regions, GAG3 and POL3, which are functional homologues of retroviral gag and pol genes, respectively. Pol3 is translated as a Gag3-Pol3 fusion protein dependent on a +1 programmed frameshift at a site with the overlap between the two genes. We show that the Ty3 frameshift frequency varie...

The budding yeast, Saccharomyces cerevisiae, provided two of the earliest analyzed examples of programmed translational frameshifting. The Ty family of retrotransposons uses +1 programmed frameshifting in the expression of their pol homologue, resulting in the production of a gag–pol fusion protein. The pol gene product encodes the enzymatic activi...

The process of protein synthesis must be sufficiently rapid and sufficiently accurate to support continued cellular growth. Failure in speed or accuracy can have dire consequences, including disease in humans. Most estimates of the accuracy come from studies of bacterial systems, principally Escherichia coli, and have involved incomplete analysis o...

A variety of cellular factors affect the movement of the retrovirus-like transposon Ty1. To identify genes involved in Ty1 virus-like particle (VLP) function, the level of the major capsid protein (Gag-p45) and its proteolytic precursor (Gag-p49p) was monitored in a subset of Ty1 cofactor mutants. Twenty-nine of 87 mutants contained alterations in...

During the process of translation, an aminoacyl tRNA is selected in the A site of the decoding center of the small subunit based on the correct codon-anticodon base pairing. Though selection is usually accurate, mutations in the ribosomal RNA and proteins and the presence of some antibiotics like streptomycin alter translational accuracy. Recent cr...

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recent...

Benzo[a]pyrene diol epoxide (BPDE), the active metabolite of benzo[a]pyrene present in tobacco smoke, is a major cancer-causing compound. To evaluate the effects of BPDE on human breast epithelial cells, we exposed an immortalized human breast cell line, MCF 10A, to BPDE and characterized the gene expression pattern. Of the differential genes expre...

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the freq...

During protein synthesis, interactions between the decoding center of the ribosome and the codon-anticodon complexes maintain translation accuracy. Correct aminoacyl-tRNAs induce the ribosome to shift into a "closed" conformation that both blocks tRNA dissociation and accelerates the process of tRNA acceptance. As part of the ribosomal recognition...

Programmed translational frameshift sites are sequences in mRNAs that promote frequent stochastic changes in translational reading frame allowing expression of alternative forms of protein products. The EST3 gene of Saccharomyces cerevisiae, encoding a subunit of telomerase, uses a programmed +1 frameshift site in its expression. We show that the s...

Estimates of missense error rates (misreading) during protein synthesis vary from 10(-3) to 10(-4) per codon. The experiments reporting these rates have measured several distinct errors using several methods and reporter systems. Variation in reported rates may reflect real differences in rates among the errors tested or in sensitivity of the repor...

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an...

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stim...

The reassignment of stop codons is common among many ciliate species. For example, Tetrahymena species recognize only UGA as a stop codon, while Euplotes species recognize only UAA and UAG as stop codons. Recent studies have shown that domain 1 of the translation termination factor eRF1 mediates stop codon recognition. While it is commonly assumed...

A previous survey of Euplotes genes available in GenBank indicated that >5% required a +1 translational frameshift for expression, much higher than observed in other organisms. Because this sample of sequences was non-random and biased towards highly expressed genes, we have randomly selected and sequenced 25 Euplotes crassus macronuclear DNA molec...

When the yeast Saccharomyces cerevisiae shifts from rapid growth on glucose to slow growth on ethanol, it undergoes profound changes in cellular metabolism, including the destruction of most of the translational machinery. We have examined the effect of this metabolic change, termed the diauxic shift, on the frequency of translational errors. Recod...

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts e...

Recent work suggests that there is a high frequency of programmed +1 translational frameshifting in ciliates of the Euplotes genus. Frequent frameshifting may have been potentiated by stop codon reassignment, which is also a feature of this group.

The ribosome is a molecular machine that converts genetic information in the form of RNA, into protein. Recent structural studies reveal a complex set of interactions between the ribosome and its ligands, mRNA and tRNA, that indicate ways in which the ribosome could avoid costly translational errors. Ribosomes must decode each successive codon accu...

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-fr...

Excerpt A commonly held belief, especially among beginningstudents of molecular biology, is that the process of protein synthesis is faithful—the sequence of codons in anmRNA defines exactly the sequence of amino acids in theencoded protein. Of course, like any biological process,protein synthesis is inherently error-prone. Nevertheless,it is impor...

The complexity of the nonsense-mediated decay (NMD) system makes it difficult to study by comparing the expression of various single reporter constructs. The known effects of the NMD genes include a reduction both in mRNA stability (reviewed by Czaplinski et al., 1999) and in the efficiency of translational initiation (Muhlrad & Parker, 1999) of no...

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficien...

The ribosome rapidly translates the information in the nucleic sequence of mRNA into the amino acid sequence of proteins. As with any biological process, translation is not completely accurate; it must compromise the antagonistic demands of increased speed and greater accuracy. Yet, reading-frame errors are especially infrequent, occurring at least...

Translational frameshifting is a ubiquitous, if rare, form of alternative decoding in which ribosomes spontaneously shift reading frames during translation elongation. In studying +1 frameshifting in Ty retrotransposons of the yeast S. cerevisiae, we previously showed that unusual P site tRNAs induce frameshifting. The frameshift-inducing tRNAs we...

Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the prog...

Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from...

According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block...

There are several ways that genes may encode alternative products. The most widely recognized mechanism is alternative splicing. However, genes may also employ noncanonical translational events to produce such products. Some of these mechanisms operate at the level of translational initiation. In prokaryotes, genes may include alternative ribosome-...

The translational apparatus very efficiently eliminates errors that would cause a spontaneous shift in frames. The probability of frameshifting can be increased dramatically by either cis or trans-acting factors. Programmed translational frameshift sites are cis-acting sequences that greatly increase the frequency of such errors, at least in part b...

Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product,...

Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product,...

Up to this point the discussion has focused on events in which the reading frame shifts between overlapping codons. However, shifts can occur in larger increments, up to as many as 50 nt. Such an event is termed a translational hop. Translational hopping was first observed in synthetic constructs made to test the sequence requirements of frameshift...

In general, the translational machinery more efficiently eliminates frameshift errors than any other sort. It is actually difficult to estimate the frequency of these errors because the system used to measure them probably introduces specific sites which stimulate frameshifting much more efficiently than at random sites in mRNAs, as discussed below...

By far the most numerous and the most ubiquitous of frameshift sites are a class of −1 frameshift sites first described in metazoan retroviruses. These sites have been found in retroviruses,27,36,40–43,56,59,63,64 coronaviruses, 5,17,18,25,35 arteriviruses,31,61 astroviruses,44,57,99 giardiaviruses,96 toroviruses,80 several plant viruses,4,47,62,73...

Subtle differences among mRNA sequences, interacting in unpredicted ways with elements of the translational apparatus, generate the continuum of potential translational frameshift sites, most of which are so inefficient as to be undetectable, but some of which either constitutively promote detectable frameshift events or can be induced to promote s...

By analogy to the characterized eukaryotic sites, putative simultaneousslippage −1 shift sites have been identified in E. colt in the chromosomal gene for two subunits of DNA polymerase III, dnaX,2,18,51 at the 3’ end of gene 10 of bacteriophage T7,8,9,14 within the overlap between the lambda phage tail genes, G and T,30 and in many insertion seque...

The nomenclature of alternative translational events betrays a prejudice concerning the mechanism by which they occur. We speak of “programmed translational frameshifting,” “programmed translational hopping” and “programmed translational readthrough.” In describing the events in this way, a distinction is drawn between these relatively efficient ev...

Arguably, the most essential process in a living cell is the translation of genetic information into protein products. Molecular genetic research has focused for the most part on the transcription of the storage structure for genetic information, DNA, into messenger RNA. It is clear that much of genetic control is exerted at this level—appropriate...

If alternative decoding events occur only during a translational pause, then any translational pause of sufficient duration could cause an aberrant event to occur. Of course, all translational pauses do not since much more is required than simply a translational pause. We have seen in the preceding chapters that a translational pause at an appropri...

Errors that alter the reading frame occur extremely rarely during translation, yet some genes have evolved sequences that efficiently induce frameshifting. These sequences, termed programmed frameshift sites, manipulate the translational apparatus to promote non-canonical decoding. Frameshifts are mechanistically diverse. Most cause a -1 shift of f...

Programmed translational frameshifting is a ubiquitous, though rare, mechanism of gene expression in prokaryotes and eukaryotes. Research on many such sites has led to a general understanding that frameshifting depends on slippage of one or two ribosome-bound tRNAs on the mRNA. We recently found an example of an efficient frameshift in the Ty3 retr...

Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation. A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tR...

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a val...

Recently we described an unusual programmed +1 frameshift event in yeast retrotransposon Ty3. Frameshifting depends on the presence of peptidyl-tRNA(AlaCGC) on the GCG codon in the ribosomal P site and on a translational pause stimulated by the slowly decoded AGU codon. Frameshifting occurs on the sequence GCG-AGU-U by out-of-frame binding of a val...

Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for...

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Most retroviruses and retrotransposons express their pol gene as a translational fusion to the upstream gag gene, often involving translational frameshifting. We describe here an unusual translational frameshift event occurring between the GAG3 and POL3 genes of the retrotransposon Ty3 of yeast. A +1 frameshift occurs within the sequence GCG AGU U...

Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites. Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer. We have demonstrated the existence of a repression site distal to the enhancer...

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes a...

Transcription of Ty1 and Ty2 retrotransposons of the yeast Saccharomyces cerevisiae is modulated by multiple downstream regulatory sites. Both transposon families include a positively acting site within the transcribed region which resembles a higher eukaryotic enhancer. We have demonstrated the existence of a repression site distal to the enhancer...

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes a...

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of...

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synt...

Ten fragments of higher eucaryotic DNA were tested for upstream activation sequence activity in Saccharomyces cerevisiae by inserting them upstream of a CYC1::lacZ promoter lacking an upstream activation sequence. Fragments containing the 21-base-pair repeat region, the enhancer of simian virus 40 or both strongly stimulated beta-galactosidase synt...

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the e...

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the e...

The Ty (transposon yeast) family of retroviral-like transposons include two genes, TYA and TYB, analogous to the gag and pol genes of metazoan retroviruses. TYB lies downstream of TYA, the two genes overlapping by 38 base pairs. The primary translation product of TYB is a TYA/TYB fusion protein whose expression has been inferred to occur by transla...

The Ty elements of yeast are a family of retrovirus-like transposons that are highly transcribed, accounting for about 10% of total mRNA. We have mapped two sites to the nontranscribed region of the element upstream of the transcription start site that are required for maximal gene expression and are similar to sites previously defined in other gen...

Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo delta insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty delta-delta transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for t...

We have determined the DNA sequence of the transposable element Ty912 of yeast. The 5918-base-pair element encodes two genes, tya912 and tyb912, which specify proteins similar to sequence-specific DNA-binding proteins of Escherichia coli and retroviral reverse transcriptases, respectively. The tyb912 gene is atypical of eukaryotic genes since (i) i...

We have determined the nucleotide sequence of the yeast HIS4 gene and its 5' and 3' flanking sequences. The protein chain has a calculated Mr-value of 87 935. Th 5' end of the HIS4 transcript maps at a position 63 bp upstream from the site of initiation of protein synthesis. The 3' end of the HIS4 transcript maps at a position approx. 118 bp after...

Five ICR-170--induced mutations at the His4 locus in yeast are +1 G.C (G, guanine; C, cytosine) additions in DNA regions that contain multiple G.C base pairs. These mutations represents both nonsuppressible and suppressible alleles. All externally, suppressible frameshift mutations occur in glycine and proline codons to produce the four-base codons...

Excerpt Recent studies on the organization of chromosomal DNA in eukaryotes demonstrate that the eukaryotic genome contains families of dispersed repetitive DNA sequences (Finnegan et al. 1978; Cameron et al. 1979). These repeated sequences are several kilobase pairs (kbp) in length and differ widely in copy number and position among different stra...

Two unstable mutations at the his4 locus of yeast are due to the insertion of the transposable elements Ty912 and Ty917 into the his4 regulatory region. The two transposons are related, one being derived from the other by a substitution of 4000 base pairs of DNA. Element Ty912 includes identical terminal repeats, whereas the terminal repeats of Ty9...

The his4-912 mutation results from insertion of a transposable element into the 5'-non-coding region of the his4 gene of yeast. A duplication of 5 base pairs of wild-type his4 DNA flanks the inserted element. The major class of His+ revertants result from excision of most of the transposable element, leaving an inserted segment of 334 base pairs fl...

140 independently occurring spontaneous mutations in the lacI gene of Escherichia coli have been examined genetically and physically. DNA sequence analysis of a genetic “hotspot” shows that the tandemly repeating sequence 5′-C-T-G-G-C-T-G-G-C-T-G-G-3′ generates mutations at a high rate, either deleting or adding one unit of four nucleotides (C-T-G-...

Yeast transformation, recently described by Hinnen et al. (1), permits the introduction of cloned DNA segments into the genome of the yeast Saccharomyces cerevisiae. The system requires a highly enriched source of yeast DNA and a reasonably stable recipient yeast strain.

The structural gene for the lac repressor of Escherichia coli, the lacI gene has been sequenced. This 1,080 base pair region of the E. coli chromosome codes for the lac repressor protein of 360 amino acids. The DNA sequence largely confirms but extends the previously reported protein sequence and allows a structural analysis of genetic phenomena.

Nonsense mutations derived from 90 different codons in the lacI gene of Escherichia coli have been correlated with the I gene nucleotide sequence. In over 80 cases the specific codon which generates the nonsense mutation can be identified. The sequence shows that 14-16 sites arise through tandem double base changes.

In the lacI gene of Escherichia coli spontaneous base substituion hotspots occur at 5-methylcytosine residues. The hotspots disappear when the respective cytosines are not methylated. We suggest that the hotspots may result from the spontaneous deamination of 5-methylcytosine to thymine, which is not excised by the enzyme DNA-uracil glycosidase.

Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from...