paper transcription and translation

The production of proteins is completedthrough two processes: transcription and translation. Each nucleotide contains a sugar, and a nitrogenous base and a phosphate group. This activity can be used to Transcription starts inside the nucleoid with formation of the RNAPpromoter complex. Molecules of tRNA bind to the codons of the mRNA at the A site before moving to the P site where their amino acid is attached to the end of the growing polypeptide chain. Zhang, Y. et al. Thank you for visiting nature.com. All strains were grown overnight in LB medium at 37C from a single colony for eYFP-T7 RNAP experiments. The analysis codes that were used in this study are available from the corresponding author upon request. d A kinetic model of in vivo transcription. In the analysis, we excluded cells having too many eYFP-T7 RNAPs, because it is difficult to determine the specific binding above the high fluorescence background. As the RNA molecule grows it is separated from the template strand. Google Scholar. Annu. From Molecules to Organisms: Structures and Processes. Ribosomes exist in the cytoplasm and rough endoplasmic reticulum of eukaryotes. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000 to 2,000 nucleotides beyond the end of the gene being transcribed. Grey, Kindred, Figure 11.7: Summary of translational initiation. Transcription is performed by enzymes called RNA polymerases, which link nucleotides to form an RNA strand (using a DNA strand as a template). Cabrera, J. E. & Jin, D. J. The change of the medium and the additional growth period without the expression of eYFP-T7 RNAP allows the maturation of the FPs already expressed in cells and the dilution of eYFP-T7 RNAP to a proper concentration in a cell for the single-protein detection. 0000001611 00000 n Splicing starts with an autocatalytic cleavage of the 5 end of the intron leading to the formation of a circular or lariatwherea 5UG sequence pairs with an internal adenine (A) or branch site. In the meantime, to ensure continued support, we are displaying the site without styles Department of Chemistry, Seoul National University, Seoul, 08826, Korea, Department of Physics, Pohang University of Science and Technology, Pohang, 37673, Korea, School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, 37673, Korea, Department of Cell and Molecular Biology, Uppsala University, 75236, Uppsala, Sweden, You can also search for this author in Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation. The information that is stored in DNA molecules is rewritten or transcribed into a new RNA molecule. Bai, L., Santangelo, T. J. The translation is the process of protein synthesis where the information on RNA is expressed in the form of polypeptide chains. DEFINITION :- Transcription is synthesis of single stranded RNA from a double stranded DNA template. Addition of a poly(A) tail. Following the formation of the pre-initiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed with the polymerase synthesizing pre-mRNA in the 5 to 3 direction. 1e, red), were similar to those of T7p_4.5kb. First, RNA polymerase binds to what is known as promoter DNA. Other regulatory elements within the promoter region will be discussed in section 12.1. The first step is transcription which produces a messenger RNA (mRNA) molecule. Sci. Translation is initiated by the assembly of the small ribosomal subunit (40S) with initiation factors (IF), which recognize the 5 cap of the mRNA. a A schematic of the gene system used to detect location of the lacZ gene locus transcribed by E. coli RNAP (left panel). Red and blue bars denote the average relative x-positions of lacZ gene loci of T7p_4.5kb and T7p_3.3kb, respectively, within each time window. ADS Campos, M. & Jacobs-Wagner, C. Cellular organization of the transfer of genetic information. Publication . The deletion of RBS site causes only 30% less movement of gene loci (Fig. The elongation rates measured by RT-PCR were 1017 and 9112bps1 for the T7p_4.5kb and T7p_3.3kb strains, respectively, which are consistent with the results of image analysis shown in Fig. 1d, e, we determined the elongation rate based on real-time reverse-transcription polymerase chain reaction (RT-PCR). These are specific locations on a chromosome that are composed of atranscribed region and a regulatory (or promoter) region. A total of 643L of cells in a fresh M9 medium was prepared; 43L of cells were withdrawn into 150L of pre-cooled RNAlater solution (Ambion, AM7020) before adding IPTG (this is the sample for 0s). Scale bar, 1m. The distribution of RNA polymerase in Escherichia coli is dynamic and sensitive to environmental cues. Transcription and translation contribute to gene locus relocation to the nucleoid periphery in, $$\overline {I_{{\mathrm{max}}}} = \overline {{\mathrm{Intensity}}\;{\mathrm{of}}\;{\mathrm{the}}\;{\mathrm{ROMIF}}} - \overline {{\mathrm{cellular}}\;{\mathrm{background}}}.$$, \(\overline {\langle{I_{{\mathrm{max}}}(t)}\rangle}\), $$I_{{\mathrm{transcribing}}}\left( t \right) = \overline {\langle{I_{{\mathrm{max}}}\left( t \right)}\rangle} - \overline {\langle{I_{{\mathrm{max}}}\left( 0 \right)}\rangle},$$, \(\overline {\langle{I_{{\mathrm{max}}}\left( 0 \right)}\rangle}\), \(\overline {\langle{I_{{\mathrm{single}}}}\rangle}\), \(\langle{I}_{{\mathrm{single}}}\rangle\), \(\langle\overline {I_{{\mathrm{single}}}}\rangle\), \(\langle{\mathrm{{RNAP}}}_{{\mathrm{transcribing}}}\rangle\), \(\frac{{I_{{\mathrm{transcribing}}}\left( t \right)}}{{\langle\overline {I_{{\mathrm{single}}}}\rangle }}.\), https://doi.org/10.1038/s41467-019-13152-y. Since it has been known that Ksg treatment leads to the change in the nucleoid morphology44, we assessed whether the nucleoid morphology before and after Ksg treatment differ and observed no change in the nucleoid morphology in our experimental conditions (Supplementary Fig. It has been reported that transcription activity of E. coli RNAP increases in the presence of the ribosomal protein S1 in vitro46. Translation Transcription that involves translation from one language to another presents an especially complex and challenging task. General properties of transcriptional time series in Escherichia coli. The distribution of the integrated fluorescence intensity (denoted as \(\langle{I}_{{\mathrm{single}}}\rangle\)) from each eYFP is shown in Supplementary Fig. Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Thus, the width of the nucleoid from the center of the cell was 300nm. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Elf, J., Li, G. W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Alternatively, RNA polymerases I and III require termination signals. Google Scholar. The tRNA in the E site will be released, and a new tRNA will enter into the A site, and the process will continue with the addition of tRNAs in the manner until the full message is transcribed (figure 11.8). Each point represents independent measurement. In transcription, adenine is paired with uracil in RNA and guanine is paired . T7 RNAP was expressed from an L-rhamnose inducible plasmid (pNL004). The process by which the mRNA codes for a particular protein is known as Translation. T7 RNAP was expressed from the plasmid pNL003 (inducer, l-rhamnose). No further modifications are required for the mRNA molecule and it is possible for translation to begin immediately. These transcriptional kinetic parameters can be derived from the appearance of fluorescent foci and an increase in their intensity, which represents the number of T7 RNAPs transcribing genes per cell as a function of time after IPTG addition (Fig. 1c). 3d). We constructed a strain expressing membrane-bound Tsr-eYFP (NL003)32 and quantified the fluorescence intensity of single eYFPs under the same imaging condition as the eYFP-T7 RNAP measurements (Supplementary Fig. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. If the researcher is not a native speaker of the language used by research participants, translators are often required. Small nuclear RNAs have a variety of functions, including splicing pre-mRNAs and regulating transcription factors. Describe the structure of mRNA, including the. The blocking of translation initiation resulted in a reduced gene locus movement of 592nm (Fig. Ht0E{}r`14AlB@ -)&z#eH;6QR* HV@9Ji(];j>D)I4w -pcQNR3.a$%9)c!W5QVoE|nU'I`8K%j\_{G%ay7 UXgvB*KB}BSRsY#ddS7mWTa&'L\'^]Llu Qst_#fb/C1Yx?AsRtm{Wgr1{.RuNnYn~Z}HNO;|d@1Rh/N! These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more active genes that are constantly being expressed by the cell. This section provides guidance on 1) budgeting time and resources required b, c Distributions of the subcellular localization of a gene locus transcribed by T7 RNAP. Microbiol. Ask an Expert. Transcription and Translation. Biol. and J.B.S. CAS 5), which denotes the locations of the lac operon without transcription (repressed condition)31. RBS-deleted strains expressed several hundred times lower levels of lacZ proteins than a control strain (Miller assays, Supplementary Fig. The data represent means.d. 350L of lysis buffer (PureLink RNA Mini Kit) containing 1% beta-mercaptoethanol (Sigma, M3148) was added to cells and vortexed for 10s. Then, total RNA was extracted following the procedures described in the quick reference of PureLink RNA Mini Kit. We blocked ribosome binding to the DNAT7RNAPmRNA complex by adding Ksg, which inhibits translation initiation44. Once at the cap, the initiation complex tracks along the mRNA in the 5 to 3 direction, searching for the AUG start codon. Commun. Two lac operators (O1) were placed up- and downstream of the T7 promoter31, which tightly suppressed expression by T7 RNAP in the absence of the inducer isopropyl--D-1-thiogalactopyranoside (IPTG) (Fig. The two main steps in gene expression are transcription and translation. Because ribosomes are typically excluded from the nucleoid43, the formation of a large DNA-RNAP-mRNA-ribosome complex may induce the movement of gene loci during transcription. The transcription occurs in the nucleus of the cell, once the DNA is copied through the replication, immediately the process of transcript formation started. Transcription and translation. However, after the addition of 1mM IPTG, which induces the dissociation of LacI from the operator sites, eYFP-T7 RNAPs were able to bind the T7 promoters and transcribe downstream of the promoters. This makes it possible for a large number of proteins to be produced from multiple RNA molecules in a short time. This is because a translator must handle the meaning of the text . Peer reviewer reports are available. The average fluorescence intensity from the transcribing eYFP-T7 RNAPs bound to DNA was obtained from: where \(\overline {\langle{I_{{\mathrm{max}}}\left( 0 \right)}\rangle}\) is the average of Imax obtained from the repressed condition, i.e., without IPTG induction (defined as time=0). As shown in Fig. Cells were collected by centrifugation at 10,000rpm, 4C for 2min. The lacZ-6xTetO strain was constructed by inserting a KanR-6xTetO fragment downstream of the lacZ gene, while lacY and lacA genes were deleted. Average relative x-positions of the gene loci from Fig. The intensity of auto-fluorescence was obtained from the cells without the plasmid pNL003, which expresses eYFP-T7 RNAP. The relocated distribution determined by the simulation matched well with the distribution shown in Fig. 1e, blue line, for convenience. Transcription and translation take the information in DNA and use it to produce proteins. Annu. a Representative images of T7p_4.5kb cells acquired late (>270s) after adding IPTG. RNA chain initiation 4. This is analogous to the pause sequences upstream of attenuation sites, where coupling controls transcription termination (). Genes provide information for building proteins. Each tRNA molecule is bound to an amino acid and delivers the necessary amino acid to the ribosome. The cells were grown for 5h with 1mM IPTG to induce lacZ expression (OD600 ~0.3). Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli. You are using a browser version with limited support for CSS. For each example: 2nd correct mRNA basesDNA by transcribing a.Fill llin inthe the complimentary strand the bottom DNA code. Replication, Transcription, and Translation - Free Paper Sample Replication, transcription, and translation are the three processes in DNA, RNA, and protein synthesis that create the variety of life found on earth. 2d (Fig. T7 RNAP expressed from a low-copy-number plasmid backbone pVS133 (pNL004). & Wang, M. D. Single-molecule analysis of RNA polymerase transcription. 6). We used Spatzcells58 in the mRNA fluorescence images to quantify the lacZ mRNA in individual cells. Each point represents independent measurement. CC BY SA 3.0. Demo, G. et al. The students will be asked to take a strand of DNA and first transcribe it. (Supplementary Fig. In this model, DNA polymers are confined in the center of the cell by conformational entropy, while multiple 70S-polysomes formed on nascent mRNA inside the nucleoid diffuse outside the nucleoid due to the excluded volume effect. Two strands of DNA are bonded together by their nitrogenous bases. Biophys. 0000003133 00000 n Science 333, 14451449 (2011). When the tRNA recognises its complementary codon in the mRNA strand, it goes to collects its specific amino acid. 3b, gray); the movement of the lacZ gene locus with the RBS was ~329nm, while nearly no movement was observed for the lacZ gene locus without the RBS (24nm) (Fig. Microbiol. Although the localization error of each dot was 70nm, the error of the averaged subcellular localization was lower than 30nm, as the number of measured foci increased to 350 at the 5% significance level (Supplementary Fig. Transcription is the first step in gene expression. The distribution following these movements is comparable with the distribution in Fig. a Schematic of the gene system used to detect the location of the lacZ gene transcribed by wild-type T7 RNAP. c],|mpt]sagD/\Nb( pp 255. PubMed 55, 601610 (2005). After centrifugation, cells were resuspended to OD600 ~0.1 with 0.4% glycerol, amino acids, vitamins, and 0.2% l-arabinose and grown for 3h to express TetR-eYFP. Dev. Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. USA 112, E4390E4399 (2015). Google Scholar. 4) DNA zips back up and the mRNA leaves the nucleus and enters the cytoplasm. DNA is a long molecule made up of many subunits, or monomers, called nucleotides. T7 RNAP has no known interaction with ribosomes and moves approximately five times faster than E. coli RNAP. RNA polymerase unwinds and separates the DNA by creating a structure known as the transcription bubble. pp 277. Thus, we analyzed the locations of the fluorescent foci in each cell over time after inducing transcription with IPTG (Fig. e Comparison of gene locus movement under translation initiation inhibition conditions. xb```"V,Ad`B&F3)wjTbbva'\lRprr9C Translation is the process by which mRNAs are converted into protein products through the interactions of mRNA, tRNA, and rRNA. Lauren Simonds. Then, 0.5L of 10% SDS solution (Sigma, L3771) dissolved in 1mL of RNase-free water (PureLink RNA Mini Kit, Invitrogen, 12183018A) was added to cells and vortexed for 10s. Cells were incubated at room temperature for 5min. Endogenous E. coli RNAP transcribes ~4000 genes. Each cell typically contained one or two foci depending on its cell cycle. 1e. Cell Biology, Genetics, and Biochemistry for Pre-Clinical Students by Rene J. LeClair is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted. As one tRNA molecule moves from the P site into the E site another tRNA molecule moves from the A site into the P site and delivers the next amino acid to the polypeptide chain. Google Scholar. Figure 14.4 A schematic view of a eukarytoic gene, and steps required to produce a protein product. Based on our results, we suggest that relocation of the gene locus by transcription occurs as follows (Fig. & Treutlein, B. Single-molecule studies of RNA polymerases. The releasefactors in both prokaryotes and eukaryotes instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. 6, e1000694 (2010). McGuffee, S. R. & Elcock, A. H. Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm. In transcription, an expert has to listen to an audio file and transcribe the spoken words in the same language on a piece of paper or on a computer file. Free floating RNA nucleotides get matched up to the DNA following the base pairing rules. Genet. ISSN 2041-1723 (online). PubMed Performance Expectations. 1c. 2017. Article To overcome the limitation of using FP-tagged endogenous E. coli RNAP, we use T7 RNAP fused with enhanced yellow fluorescent protein (eYFP) and inserted one copy of the T7 RNAP-specific promotor in the E. coli genome34. Google Scholar. The amino acid attached to the tRNA in the P site will be transferred to the tRNA in the A site; this is referred to as the peptidyl transferase react ion. Science 338, 9497 (2012). 1e, blue) and obtained kon (0.460.03min1, meanstandard deviation) and koff (1.120.11min1). Indeed, LacZ mRNA with the RBS was expressed at a 36-fold higher level relative to LacZ mRNA expression in the RBS-deleted strain (Supplementary Fig. & Nudler, E. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Bakshi, S., Choi, H., Mondal, J. 4d). Translation: On the worksheet, make the mRNA codons into tRNA codons (review Transcription to Protein Synthesis sheet). Alternatively, high RNAP activity would dissociate NAPs from DNA, maintaining the bacterial chromosomal structure, which may also change the looping and local DNA structure. Choose your answers to the questions and click 'Next' to see the next set of questions. J. Bacteriol. Much of the knowledge of transcription has been derived from in vitro studies, in which purified RNA polymerases (RNAPs) and relatively short DNAs have been used at dilute concentrations in isolation from other machinery proteins. Cell and Molecular Biology: Concepts and Experiments, 7th ed. As a result, gene movements during E. coli RNAP transcription (3050nm) and T7 RNAP transcription (6080nm) correspond to 1016% and 2027% of the nucleoid size (width), respectively. A home-built software (Matlab) was used for image analysis. The specific sequence provides the information for the production of a specific protein. Transcription is the process of producing a strand of RNA from a strand of DNA. Its produces messenger RNA ( mRNA). The copying of DNA to mRNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every complementary nucleotide read in the DNA strand. 3e, blue bars). & Studier, F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. The major steps of transcription are initiation, promoter clearance, elongation, and termination. The deletion of the RBS from the lacZ gene (T7p_4.5kbRBS and T7p_3.3kbRBS) also resulted in an ~35% shorter gene locus movement in both strains (Fig. The mCherry-6xTetO strain was constructed by replacing the lacZ gene with the mCherry gene using lambda red recombination and then a KanR-6xTetO fragment was inserted into the downstream of the mCherry gene. 12). Each point represents independent measurement. 5d after a 15-min incubation with 5mgmL1 kasugamycin (Ksg), which inhibits translation initiation. To remove the effects of direct interactions between RNAPs and ribosomes15,16,17 and quantify only the effect of ribosome binding to mRNAs to the degree of gene loci movement, we used T7 RNAP-driven transcription for this analysis (T7p_lacZ-12xTetO). 3a). <]>> Transcription and Translation Model by Andrew Scott 5.0 (5) $3.00 Word Document File This is a template for a model that allows students to physically follow the process of transcription and translation. Translation ends when a stop codon on the mRNA strand reaches the A site in the ribosome. Lieberman M, Peet A. The average number of transcribing T7 RNAP (denoted as \(\langle{\mathrm{{RNAP}}}_{{\mathrm{transcribing}}}\rangle\)) was obtained by \(\frac{{I_{{\mathrm{transcribing}}}\left( t \right)}}{{\langle\overline {I_{{\mathrm{single}}}}\rangle }}.\). Then, cells were incubated in 2mL of fresh M9 medium supplemented with 0.4% glucose and 5mgmL1 Ksg (Sigma-Aldrich, #32354). & Weisshaar, J. C. Entropy-based mechanism of ribosome-nucleoid segregation in E. coli cells. Added Myoglobin by AzaToth. Instead, a protein called a release factor binds to the stop codon and adds a water molecule to the polypeptide chain when it moves into the P site. H.J.A. Only one strand of a DNA double helix is transcribed for each gene. Translation can begin in bacteria while transcription is still occurring. https://archive.org/details/11.6_20210926. 1). 4.9. The RNAPpromoter complex forms inside the nucleoid. What are some similarities between transcription and DNA replication? c, d Distributions of the subcellular localization of transcribing eYFP-T7 RNAP foci along the short axis. This process is known as DNA replication. 3d. helped measurements and analysis. Grey, Kindred, Figure 11.8 Summary of translational elongation. Further information on research design is available in theNature Research Reporting Summary linked to this article. Transcription and translation are the two processes that convert a sequence of nucleotides from DNA into a sequence of amino acids to build the desired protein. The section of DNA or the gene that is transcribed is known as the transcription unit. Elife 6, 117 (2017). First-strand cDNAs were synthesized using Superscript III Reverse Transcriptase (Invitrogen, 18180-044) and RNaseOut Recombinant Ribonuclease Inhibitor (Invitrogen, 10777-019). b, c Representative images of eYFP-T7 RNAPs in E. coli cells. 8). 1, 2, 57, and 915 are provided as a Source Data file. Thus, the subcellular positions of eYFP-T7 RNAP foci were expected to be mainly localized at the central region of the cell. We measured the mRNA expression level of E. coli RNAP-driven strains using RT-PCR (Supplementary Fig. The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. PubMed Central Karp, G., and J. G. Patton. It is well known that transcription accumulates positive supercoiling ahead of RNAP and negative supercoiling behind RNAP53. After the pre-class assignments you should be able to: Discuss the four levels of protein structure and relate them to the function of a protein Define the term "denatured" as it relates to protein structure By the time you take the second midterm you . The size of the nucleoids was maintained for the various measurements using both T7 RNAP and E. coli RNAP (short axis=0.600.01m). This DNA is a sequence that signals the start of genetic information for a particular gene. The run method contained a holding stage with initial heating at 50C for 2min and an initial denaturation step at 95C for 10min, followed by a cycling stage for 40 cycles with a denaturation step at 95C for 15s and an annealing/amplification step at 60C for 1min. 4b). tRNA primarily functions to bring amino acids to the ribosome during protein translation. 0000005503 00000 n We fit the data of T7p_4.5kb with a 4.5-kbp gene length using the kinetic model (Fig. The time-dependent averaged locations of the fluorescent foci were plotted after transcription was induced with 1mM IPTG (Fig. PubMed The translation to protein is more complex because groups of three mRNA nucleotides . 2021. https://archive.org/details/11.6_20210926. g The spots obtained by the simulation in Fig. The localized TetR-eYFP foci without the 6xTetO site in the genome were not observed (Supplementary Fig. We show that the subcellular relocation of gene loci is a general phenomenon that occurs during transcription by both E. coli RNAP and T7 RNAP in E. coli cells. Likewise, the polypeptide chain may be split into smaller chains or have amino acids removed. Golding, I., Paulsson, J., Zawilski, S. M. & Cox, E. C. Real-time kinetics of gene activity in individual bacteria. In a nutshell, transcription is the process of constructing mRNA from a gene with the help of RNA polymerase. The first step of gene expression is called transcription. Google Scholar. 4. A system is described which permits the efficient synthesis of proteins in vitro at high temperature based on the use of an unfractionated cell lysate from Sulfolobus solfataricus previously well characterized in the laboratory for translation of pretranscribed mRNAs, and now adapted to perform coupled transcription and translation. Mol. 1c indicate the locations of genes actively transcribed by T7 RNAPs, we measured the elongation kinetics of T7 RNAP using fluorescent imaging. performed the simulation. Students who demonstrate understanding can: Construct an explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells. The lacZ gene locus moved to the nucleoid periphery after IPTG induction (lacZ-6xTetO strain, blue squares) (>1500 spots). The introns are removed by a process called splicing to produce messenger RNA (mRNA)and the ends of the RNA molecule are processed. For the construction of T7p_3.3kb and T7p_3.3kbRBS strains, we put a Tpi-KanR fragment downstream of the lacZ gene. Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K. & Lia, G. Single-molecule approach to molecular biology in living bacterial cells. These combine to make the full 80S ribosome required for protein translation. 11), which was not observed in other experimental conditions that expressed less amounts of mRNAs compared with T7p_lacZ-12xTetO strain. The strand of mRNA moves through the ribosome from the A site to the P site and exits at the E site. Thus, it is possible that the transertion of LacY, i.e., the cotranscriptional translation and simultaneous insertion of membrane proteins in the plasma membrane42, causes repositioning of the chromosomal locus. Amino Acid Chains: Using the Genetic Code chart, fill in the amino acids for each DNA strand. Then, the normalized relative position of each dot was determined.