High-Resolution View of Bacteriophage Lambda Gene Expression by Ribosome Proﬁling

High-resolution view of bacteriophage lambda gene expression by ribosome proﬁling

Xiaoqiu Liua,1, Huifeng Jiangb,1,2, Zhenglong Gub, and Jeffrey W. Robertsa,3

aDepartment of Molecular Biology and Genetics and bDivision of Nutritional Sciences, Cornell University, Ithaca, NY 14853

Contributed by Jeffrey W. Roberts, May 23, 2013 (sent for review April 19, 2013)

Bacteriophage lambda is one of the most extensively studied strong apparent translation pauses within coding segments (10). organisms and has been a primary model for understanding basic There frequently are clusters over translation initiation and modes of genetic regulation. Here, we examine the progress of termination sites, reflecting slow steps at these stages of trans- lambda gene expression during phage development by ribosome lation. As reported by Li et al. (10) and discussed further below, – profiling and, thereby, provide a very-high-resolution view of pauses are associated with upstream Shine Dalgarno sequences. lambda gene expression. The known genes are expressed in a pre- The general expression pattern matches in good detail expect- dictable fashion, authenticating the analysis. However, many pre- ations from decades of detailed study of lambda gene regulation viously unappreciated potential open reading frames become (Fig. 1). All known lambda genes and previously annotated ORFs apparent in the expression analysis, revealing an unexpected are expressed during lambda development (except orf206b and probably NP_59778.1), as are the newly identified translated complexity in the pattern of lambda gene function. ORFs that we report below.

acteriophage lambda is an original and exemplar organism The Uninduced Lysogen. In the uninduced lambda lysogen, cI, rexA, Bthat has guided the discovery of basic genetic regulatory pro- rexB, lom, and bor are the major bacteriophage genes being cesses, including transcription repression, activation, and anti- translated, in agreement with expectation (Dataset S1). Others termination (1, 2). Lambda has provided an important model to appear significantly over background, including the early genes understand the interaction of a virus with its host. Programs of ea8.5 and ea59 (both of unknown function), and the immediate lambda gene expression that establish and maintain the prophage early genes N and cro; the latter presumably become transcribed state, and that mediate the lytic pathway of growth upon infection when repression occasionally fails, although there clearly is not or induction, are well understood in terms of the basic biochemical enough expression of gene N to allow expression of most of the mechanisms and the timing of regulatory protein function. Both delayed early set of genes that depend on the gene N transcription the classic, focused analysis of gene expression and the modern antiterminator. A few other genes appear detectably over back- global approaches such as array hybridization (3, 4) have been ground (Dataset S1). Two array experiments (3, 4) measuring applied to elucidate the complex pattern of gene function during RNA agreed in identifying in uninduced lysogens most of the set lambda growth. A recent method, ribosome profiling, provides of five that we find, but each report found other genes significantly a further detailed and precise view of gene expression by capturing expressed that we do not find and, in fact, there was little the instantaneous translation sites of all of the ribosomes in a cell agreement between the two array studies about these others. (5–8). We have applied ribosome profiling to the process of lytic Differences with our measurements presumably reflect the fact growth of bacteriophage lambda to map in detail the expression that RNAs are not uniformly translated, in addition to uncer- of lambda proteins and to infer unique loci of translation. We, of tainties of array measurements. course, confirm the known patterns of gene expression, but we also expand understanding in several ways, including a complete cat- Gene Expression During Lytic Growth. After repression is relieved, alog of gene expression, the discovery of unique functional lambda gene expression occurs in two waves (11, 12). De- open reading frames (ORFs), and the discovery of bacterial repression enables promoters pL and pR to function, providing genes expressed during phage development. expression of genes N and cro at the earliest time; these genes are the only two lytic genes highly expressed at 2 min after de- Results and Discussion repression (Fig. 2 A and B). N is a transcription antiterminator Overview of Method and Approach. We chose temperature in- that potentiates transcription of the early genes to the right of N duction of the classic cI857 repressor mutant of lambda in a ly- and cro. Early genes are expressed increasingly from 5 to 10 min, sogen of Escherichia coli MG1655 to synchronize the lytic process, and then less at 20 min (Fig. 2 A and B). The decrease in early sampling the lysogen and control nonlysogen both before and 2, 5, gene expression in the last interval is attributed to the activity of 10, and 20 min after shifting the temperature from 32 °C to 42 °C. the lytic repressor Cro, which represses both pL and pR as its The last sample time was chosen to be before any significant cell concentration builds up in the cell (13). The last of the early genes lysis, but during the later stages of lytic gene expression. Se- on the right is Q, which encodes an antiterminator that provides quencing produced ∼106 ribosome prints per sample, which were expression of all of the late genes (14). Late gene expression only appears significantly at 10 min, and then increases greatly by mapped onto both phage and bacterial genomes and visualized in fl the Gbrowse genome browser at EcoWiki (9). Total protected 20 min (Fig. 2C), re ecting its dominance in the last period of nucleotides within ORFs were summed to determine the density of translation of each reading frame (5). We take this number to indicate the overall rate of translation, although obviously we are Author contributions: X.L. and J.W.R. designed research; X.L. performed research; X.L., H.J., assuming that pauses in translation do not excessively affect the and Z.G. analyzed data; and X.L. and J.W.R. wrote the paper. overall rate. Because the expression level is not normalized for the The authors declare no conflict of interest. copy number of the replicating phage DNA, it thus encompasses Data deposition: The data reported in this paper have been deposited in the Gene Ex- both the effect of DNA template availability on mRNA synthesis pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE47509). and the efficiency of utilization of messengers by ribosomes. 1X.L. and H.J. contributed equally to this work. 2Present address: Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sci- General Features of the Translation Pattern. As shown in more ences, 32 XiQiDao,Tianjin Airport Economic Park, Tianjin 300308, China. detail below, the pattern of ribosome occupancy over individual 3To whom correspondence should be addressed. E-mail: [email protected]. protein coding sequences follows that previously reported, dis- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. playing highly asymmetric distributions of protected sites due to 1073/pnas.1309739110/-/DCSupplemental.

11928–11933 | PNAS | July 16, 2013 | vol. 110 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1309739110 Downloaded by guest on September 23, 2021 Fig. 1. An overview of gene expression measured by ribosome proﬁling across the lambda phage genome during lambda prophage induction. Bar plots centered on dashed line circles from inside to outside show expression levels by ribosome proﬁling at different times after shifting the culture to 42 °C: blue, 0 min; cyan, 2 min; green, 5 min; orange, 10 min; and red, 20 min. Annotated genes of lambda are shown on the outside. At each time point, the reads per kilobase per million (RPKM) for lambda genes was normalized by scaling between 0 and 1, with 531 being the maximum at 0 min and 9,091 at 2 min. Because the RPKM values for some genes at 5, 10, and 20 min are too high, the maximum values were set at 10,000, where RPKM is reads per kilo- GENETICS base of coding sequence per million mapped reads, as originally denoted (31).

lambda gene expression as phage structural proteins accumulate. Frameshifting. A frameshift occurs between lambda genes G and Fig. S1 and Dataset S1 document in more detail the patterns of T, resulting in a fusion protein between these reading frames early and late gene expression, including translation of newly when approximately 3.5% of the ribosomes slip back a nucleo- recognized ORFs in both periods. tide at the “slippery” sequence 5′-GGGAAAG-3′ near the end Previous work has shown that although transcription across the of the G reading frame (19). There is a distinct concentration of lambda late gene region is approximately uniform, reflecting the ribosomes over this sequence, and at the normal termination single mRNA synthesized for the late genes under influence of codon of the G reading frame, which is evident upon examining the gene Q antiterminator, the yields of various proteins from the the profiles (Fig. 2F). A typical protected RNA segment covering late gene transcript are very different (15, 16). This differential the slippery sequence (in bold) is TCTGCGGGAAAGTGTTC- translation of late genes is apparent in the ribosome profile (Fig. 2 GACGGT, and a typical segment covering the termination co- C and D). It is of interest to compare the instantaneous rate of don of G is TCGAGGGTGAGCTGAGTTTTGCCCT. expression of each transcript to a measure of the ultimate re- quirement for each structural protein in the phage particle (2, 17). Translation of a Regulatory RNA. Regulation of late gene expression Fig. 2E shows some correlation, in particular for the two most in lambda and related phages occurs through transcription anti- abundant proteins D and E, and for some but not others of the termination, by the product of gene Q, of a constitutive transcript least abundant proteins. It is noteworthy that rexB, a gene from the late gene promoter (14); in lambda, this RNA (lambda 6S expressed in the prophage, shows increased expression at late RNA, not to be confused with the cellular 6S RNA) is 200 nt long. times, consistent with previous genetic evidence (18). rexB is An ORF, orf-64, begins within this RNA and extends through the transcribed from the independent promoter pLIT, and it is likely transcription terminator. orf-64 shows ribosome-protected frag- that the enhanced expression of rexB at late times is due to in- ments at a low but significant level, ∼5% the level of the adjacent creased gene dosage as phage DNA is replicated. Q gene and ∼10% the level of gene S, the first late gene regulated by Q (Fig. 3A). Although it is clear from biochemical analysis with The Pattern of Ribosome Progression. As reported (10), the distri- purified proteins that the Q protein and transcription elongation bution of ribosomes across reading frames is highly heteroge- factor NusA suffice to cause antitermination of transcription at the neous, displaying strong pause sites that are correlated both terminator of 6S RNA (14), the occurrence of concurrent trans- with sites of initiation and termination, and with internal Shine– lation in the cell could modulate or enhance the antitermination Dalgarno sequences that presumably stall ribosomes by annealing process. It may be significant that a cluster of ribosomes appears with the end of 16S RNA, as in initiation. To illustrate the general over the upstream half of the intrinsic transcription terminator pattern of translation, Fig. 2D shows a display of the distribution stem, a configuration that would inhibit termination. Such struc- of ribosomes over a segment of approximately 11 kB of the late ture is reminiscent of attenuation control in bacterial operons (20) genes. Note that the scales are drastically different for displays of and could suggest a distant evolutionary relationship between the different time samples in Fig. 2D; thus, there is a difference of these regulatory mechanisms. ∼100 in scale between 5 and 20 min, but the overall patterns are quite similar. In addition to internal pause sites, there frequently Unique Translated ORFs. A notable feature of the ribosome profiles are collections of ribosomes over initiation and termination is the abundance of translation in regions of the genome without codons of the reading frame, as noted (10). Fig. S2 uses the characterized genes but in potential ORFs of unknown function, analysis of Li et al. (10) to confirm that ribosomes in the coding many of which have not been annotated (Dataset S2 and Fig. S3). sequence tend to pause where Shine–Dalgarno sequences are We discuss below selected instances of such translation that seem positioned upstream to interact with the ribosome. to be noteworthy. However, we also attempted to catalog all such

Liu et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 11929 Downloaded by guest on September 23, 2021 Fig. 2. Expression patterns of the lambda genome from ribosome profiling. (A–C) Gene expression pattern in the early left operon (A), early right operon (B), and late gene operon (C) at different times after induction of a lambda prophage. Reads were summed over each reading frame. (D) Higher resolution view of the ribosome occupancy profile for some late genes during lambda prophage induction. (E) Correlation of gene expression levels for phage structural genes with the protein copy numbers in the purified lambda particle. (F) Ribosome occupancy profile at the translational frameshift region of lambda genes G and T. The red dashed rectangle indicates the frameshift site. Ribosomes are stalled at the slippery sequence GGGAAAG in the G reading frame; a few of these signals (approximately 3.5%) shift back one base on the mRNA into the T reading frame and continue to the end of T (19). A cluster of ribosomes over the G termination codon also is apparent.

potential ORFs in a systematic way: Dataset S2 lists 55 potential could include candidates for regions of important translation ac- ORFs distinct from known genes and previously annotated ORFs, tivity, including potential active polypeptide products. all of which display significant translation over background. This We first consider potential translated ORFs that are coded in list was obtained by (i) computing all potential ORFs greater than the predominant direction of known transcription. In several 5 aa in length with either ATG or GTG initiation and a termination cases, these ORFs occur between genes of the late region when — codon (∼3,000); (ii) removing all ORFs with apparent translation there is space a rare occurrence, because throughout the late levels below a chosen background; and (iii) removing from this list genes, a termination codon generally meshes closely with or even previously recognized genes or ORFs, and completely overlapping overlaps the next initiation codon (2). In one case, 150 bp sep- ORFs, leaving 55. Some of these potential ORFs display significant arates the termination of lambda tail gene L from the initiation upstream matches to the Shine–Dalgarno initiation sequence, but of tail gene K; an ORF of 76 amino acids (ORF 322) starts 6 bp after the termination of L and proceeds through the first 11 many do not (Dataset S2); however, this feature is not a re- fi amino acids of K (Fig. 3B). Juhala et al. (21) noted that this ORF quirement for signi cant translation. is homologous to the beginning of the counterpart of gene K in There is no evidence that any of these ORFs represent func- the lambda-related phage HK022, and they suggested that tional genes. Furthermore, we cannot rule out the possibility that fl a frameshift mutation might have severed a gene ancestral to at least some of the signal, particularly the weakest, re ects an lambda K. Our data shows that ribosome occupancy of ORF 322, entirely irrelevant background association of RNA with ribo- largely clustered just after the initiation codon, is comparable to somes. Many potential ORFs are from regions of the genome that of adjacent genes. Because it is expected that transcription “ ” “ ” designated inessential, such as the b2 region and the segment of the late gene region is uniform, this result means that the between bor and the right end (2) (Fig. S3). It is clear, however, translation activity of ORF 322 is significant. Possibly ORF 322 that inessential in the laboratory context does not mean useless in and lambda K together constitute the active protein that corre- nature. Furthermore, the level of expression of many of the po- sponds to the related protein of HK022. tential ORFs in Dataset S2 is comparable to that of known genes In a second case, found at the beginning of the late gene (as we describe below), consistent with the notion that this list transcript, there is an interval of 375 bp between the end of the

11930 | www.pnas.org/cgi/doi/10.1073/pnas.1309739110 Liu et al. Downloaded by guest on September 23, 2021 Fig. 3. Examples of expressed unique ORFs in the prevailing direction of transcription. (A) Ribosome occupancy profiles downstream of gene Q, with 20 min of induction time. Some ribosome occupancy was found on orf-64 (red), which extends beyond the 194-nt pR’ transcript that is terminated (in the absence of the gene Q antiterminator) at nucleotide 44780. The sequence of the terminator is shown, including the hairpin (red) and poly U (underlined) sequence at the end. Two other unique ORFs (ORF 915 and ORF 916) in this region also showed significant ribosome occupancy. (B) Ribosome occupancy profiles between genes L and K, including ORF 322, with 20 min of induction time. (C) Ribosome occupancy profiles between ea22 and orf61, with 20 min of induction time.

previously recognized orf-64 and the first nucleotide of gene S, for expression of the distal parts of the interrupted stf gene, but the first recognized late gene (2). Two additional ORFs occur in no evidence that it has any significance. this interval: ORF 915 (of 15 aa) and ORF 916 (of 12 aa). The Between the leftward genes exo and Ea22, there is an interval density of translation over ORF 916 is approximately one-half of 953 bp with several previously recognized leftward-directed that of gene S (Fig. 3A and Dataset S2). ORFs (orf60a, orf63, orf61), as well as orf73 (also ORF 2310 in Dataset S2) identified with a function named bin (23). We

The inessential lambda late gene stf is interrupted by a muta- GENETICS identify another, ORF 2313 (31 aa), which is shown in Fig. 3C, tion in laboratory strains, resulting in an N-terminal fragment which is expressed comparably to the known genes like exo encoded by orf-401, and a potential C-terminal fragment encoded and Ea22. by orf-314 (22). orf-401 is translated at a level comparable to In several cases, there are translated ORFs in regions previously some late genes, and orf-314 is translated much more weakly. known to be transcribed from both strands. Thus, in the region of One of two potential ORFs between the fragments (ORF 438) predominant leftward transcription to the left of gene N, the gene appears to be signiﬁcantly translated. Thus, there is the potential sieB is transcribed rightward (Fig. 4A). Mostly overlapping sieB is

Fig. 4. Unique translated ORFs in regions of overlapping or predominant opposite strand transcription. (A) Ribosome occupancy profiles downstream of gene N, with 20 min of induction time. Unique ORFs in this region are shown in green; most are antisense to gene sieB, but in the direction of prevailing transcription from pL.(B) Ribosome occupancy profiles of eight unique ORFs at the end of lambda genome, with 20 min of induction time; these ORFs are antisense to bor and lambda p78 (NP_597781.1) but in the direction of the Q-dependent late gene transcription. (C) Ribosome occupancy profiles of plus strand ORFs in the b2 region, with 20 min of induction time. The 14 ORFs shown are antisense to ea47, ea31, and ea59, but in the same direction as Q-dependent late gene transcription, which may thus converge with the N-dependent transcription from pL.

Liu et al. PNAS | July 16, 2013 | vol. 110 | no. 29 | 11931 Downloaded by guest on September 23, 2021 Fig. 5. Effect of phage induction on E. coli protein synthesis. (A) Increase in E. coli gene translation around the attB site due to escape synthesis. Time at 42 °C: blue, 0 min; cyan, 2 min; green, 5 min; orange, 10 min; and red, 20 min. (B) E. coli genes with functions likely related to lambda phage growth and signiﬁcantly up-regulated during prophage induction. (C) E. coli genes mostly with known functions not obviously related to lambda phage growth but signiﬁcantly up-regulated during prophage induction. (D) Phage induction leads to a decrease in ribosome occupancy on E. coli genes, and approximately 30% of total reads map to the phage genome at 20 min. (E) Decrease of E. coli gene translation during lambda induction.

a set of substantially translated ORFs, all oriented leftward, and through ribosome profiling of annotated E. coli genes, in the presumably derived from the N-dependent leftward transcription: same experiments described above. We find that although ap- ORF2423 (24 aa), ORF 3070 (21 aa), ORF 2426 (20 aa), ORF2429 proximately 1,000 genes are down-regulated during the 20 min of (37 aa), ORF2432 (30 aa), ORF2433 (26 aa), and ORF2434 (15 aa). induction, a substantial number, 120, are up-regulated (Dataset Between genes RZ and Nu1 of the rightward-directed late S3). Most of these changes occur primarily late in induction. transcript, there is an interval of 2,278 bp that contains the left- Approximately one-half of the up-regulated gene expression ward-directed gene bor and extends to the cos DNA packaging has a relatively trivial origin: Genes that surround the attach- site (Fig. 4B). This interval contains the rightward-directed ORF ment site attB are replicated along with the prophage (“escape 948 (45 aa), ORF 950 (35 aa), ORF 958 (31 aa), ORF 966 (27 aa), replication”) and are overexpressed primarily because of the ORF 1830 (24 aa), ORF 975 (9 aa), ORF 976 (28 aa), and ORF increased gene copy number (Fig. 5A). This enhanced expression 1832 (33 aa). Most of these display substantial translation. occurs in a region of 300–400 kB surrounding the prophage in- There is a concentration of translated ORFs directed counter tegration site (4). No down-regulated genes were found in this to the prevailing direction of transcription in the “b2” region. This region. One well-characterized set of up-regulated genes is the substantial region of approximately 5 kb, which can be deleted nearby gal operon, overexpression of which depends on both without impairing growth (2) (although prophage integration overreplication and gene N-dependent antiterminated tran- is compromised), contains three well-recognized “early” genes, scription from the phage pL promoter (4). Transcription of gal ea59, ea31, and ea47; all are directed leftward and presumably operon genes is enhanced approximately 13-fold in induced expressed primarily via gene N antiterminator-influenced left- lysogens relative to nonlysogens (4); we found that ribosome ward transcription from the early promoter pL.(ea59 is also sig- occupancy of the gal operon genes galE, galT, and galK increased nificantly expressed in the lysogen, so that another promoter also approximately 13–16-fold, in good agreement (Dataset S3). may be present.) Several translated ORFs, ORF 467 (53 aa), A prominent group of up-regulated genes located far from attB ORF 472 (14 aa), ORF 486 (14 aa), ORF 488 (17 aa), ORF 494 and, thus, stimulated by some metabolic change in the cells, is the (28 aa), and ORF496 (36 aa) are all within ea47 and are directed set of SOS DNA damage genes (examples in Fig. 5B)(24).Because rightward (Fig. 4C). Several rightward-directed ORFs, ORF 499 our induction protocol used temperature inactivation of phage (of 21 aa), ORF 1509 (of 5 aa), ORF 503 (of 17 aa), ORF 1510 (of repressor and not explicit DNA damage (e.g., UV irradiation), the 39 aa), and ORF 507 (of 7 aa), are in the 400-bp intergenic region induction of SOS genes must be due to some aberration in DNA between genes ea47 and ea31 and are expressed comparably to metabolism that arises during phage growth, likely resulting from the three leftward genes (Fig. 4C). Within ea31 are two rightward- the replication of phage DNA; this anomaly might be the accu- directed ORFs (Fig. 4C). Remarkably, ORF 511 is 73 aa long, and mulation of single-stranded regions and incompletely replicated ORF 1522 is 30 aa long. Both are translated comparably to the phage chromosomes, for example. It is noteworthy that increased adjacent leftward genes ea31 and ea59. translation of the SOS repressor LexA occurs along with that of the There also are rightward-directed ORFs within the early re- other SOS genes, although the concentration of LexA must be gion to the right of the attachment site (not illustrated). Thus, diminished. This result is in fact as expected, because LexA is self- within ea8.5, the overlapping ORFs 585 (of 130 aa) and 591 (of regulated but also rapidly degraded while the SOS-inducing signal 41 aa) show substantial translation. is active, a process that overcomes the increased expression due to The source of the transcription for these newly recognized derepression. It is also consistent with these proposals that SOS rightward ORFs is unknown. It may of course be the gene Q gene induction occurs late rather than early in induction. antiterminator-influenced late gene transcription, which is ex- Several other sets of up-regulated genes presumably respond to pected to proceed efficiently at least through the tail fiber genes the altered cellular conditions of phage growth. Induction of stf and tfa, and could easily extend well into the early region, al- subunits of ribonucleotide reductase genes may reflect increased though its efficiency likely would be reduced through collision need for dNTPs. The set of genes involved in phosphate metab- with the predominant leftward-directed N-protein-influenced olism and transport responds to cellular phosphate limitation, transcription from promoter pL. which may occur in the induced cell; the involvement of pho genes A more detailed display of expression data of some potential in lambda growth has been noted (25). The genes cpxP, htpX,and ORFs in shown in Fig. S4. degP are activated by the two component system encoded by cpxR/ A, which senses envelope stress (26), a plausible response to such Changes in Host Gene Expression After Lambda Induction. Expres- phage proteins as the lysis set that are made at late times of sion in the host was measured before and after lambda induction induction.

11932 | www.pnas.org/cgi/doi/10.1073/pnas.1309739110 Liu et al. Downloaded by guest on September 23, 2021 Finally, there is a set of up-regulated genes that mostly have product itself. Thus, translation of ORFs upstream of expressed known function, but no obvious relation to phage development; genes has a well-known regulatory function in yeast (28), and the examples are shown in Fig. 5C. very act of ribosome binding can change the structure and avail- Phage growth substantially engages the protein production ca- ability of mRNA for translation of other parts of the message, or pacity of the cell. At 20 min after induction, about 30% of the total for other functions of RNA such as transcription termination. A ribosome-protected RNA reads map to the lambda genome. Of the specific example of an alternate consequence of ribosome function 1,000 down-regulated genes (Datasets S4 and S5), 600 are down- is the expression of the lambda bar “genes,” which act to sequester regulated more than twofold. There appears to be a constancy of ribosomes that must be freed by a peptide hydrolase (29). Another total protein synthetic capacity when the number of ribosome reads possibility is that engagement of RNA with ribosomes serves to from phage and host are summed throughout the 20-min period of prevent deleterious activity (e.g., forming R loops) of free RNA phage development; the 30% increase in phage reads is matched by (30), which might be prevalent where antiterminators prevent Rho a 30% loss in host reads (Fig. 5 D and E), and the decrease in each function, as in most lambda transcription; thus, functional ORFs individual amino acid incorporation into bacterial protein is equal to its incorporation into phage proteins (Fig. S5). might be retained in regions of the genome where transcription occurs, even if no functional polypeptide is produced. Summary and Further Discussion Finally, it is of course possible that some or all of these ORFs have no function at all, representing just background activity of Ribosome profiling provides a detailed view of gene expression of both phage and host during the development of bacteriophage the transcription and translation systems. We cannot eliminate lambda. This analysis confirms the expected general patterns of the possibility that there is adventitious and nonfunctional as- gene expression but also shows an unexpected complexity of the sociation of RNA with ribosomes that gives rise to illusory translation landscape. The major finding is that much ribosome translation of at least some of these potential ORFs. Further occupancy occurs in ORFs of unknown function. Although some informatic and directed analysis, and comparative phage geno- of these potential ORFs were known from analysis of the lambda mic analysis, would be required to query the function of this genome sequence, and all could of course be inferred by simple unexpected translation activity. analysis, the translation data focuses attention on a substantial number that were not previously known to be of interest. Materials and Methods Numerous translated ORFs are large enough plausibly to The strain used in these experiments was E. coli K12 MG1655 (obtained from encode functional proteins, in the range of 20–130 aa. The un- F. Blattner, University of Wisconsin, Madison, WI), made lysogenic for λcI857 GENETICS derappreciated importance of small proteins has been recog- (from M. Gottesman, Columbia University Medical School, New York). Ri- nized, and they have a variety of roles that might be relevant to bosome protected RNAs were prepared as described (6), with minor mod- lysogen and phage growth, acting as intercell signaling factors, ifications. Detailed protocols and methods of data analysis are provided in toxins, and membrane components (27). Few of the newly dis- SI Materials and Methods. covered ORFs appear to be expressed in the lysogen at levels similar to well known prophage-specific genes (cI, rexA, rexB, ACKNOWLEDGMENTS. We thank Ryland Young and Jim Hu (Texas A&M lom, bor), although the previously recognized ORFs ea59 and University) for careful and thoughtful reading of the manuscript, Sherwood ea8.5 are significantly expressed. However, very low expression Casjens (University of Utah) for helpful comments, and Nick Ingolia for providing a protocol before publication. Help with the display and analysis of might well be important. the data was provided by the PortEco project through National Institute of Of course, the act of ribosome engagement or ribosome syn- General Medical Sciences U24GM088849, and this work was supported by thetic activity could be important rather than the translation National Institutes of Health Grant GM 21941.

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