Genomics to proteomics

1. Harini Chandra Affiliations Genomics to proteomics The genome represents only the starting point towards understanding complexity of biological functions. The products of gene expression, proteins, provide a much more meaningful insight into the mysteries of essential biological processes.

2. Master Layout 1 2 2. Single gene, multiple proteins 3. Post-translational modification of proteins 3 1. Genomic DNA contains large stretches of non-coding regions 4 Action Description of the action Audio Narration The figure above must be shown with each of the headings appearing. The headings in red are the three tabs for each animation described in the subsequent slides. The user must be allowed to click on these tabs which must lead to the corresponding animation for that heading. When the user moves the cursor over any of the remaining marked regions, the definition for that part must popup as given in the next slide. Show the figure above along with the headings which the user must be able to click on. The genome represents only the starting point towards understanding complexity of biological functions. The products of gene expression, proteins, provide a much more meaningful insight into the mysteries of essential biological processes. The number of proteins expressed in a cell is much larger than its genomic counterpart due to the post-transcriptional and post-translational modifications taking place. To obtain a clear understanding of cellular processes & regulation, there has been a gradual shift in focus from the genome to the proteome due to the large amount of information available from the proteome. 5

3. Definitions of the components:Master layout 1 1. Genome: The entire sequence of an organism’s hereditary information, including both coding and non-coding regions, encoded in DNA is known as the genome. 2. Transcriptome: The set of all RNA molecules, including mRNA, rRNA and tRNA, present in an organism is referred to as the transcriptome. 3. Proteome: The entire complement of proteins expressed by the genome of an organism under specific defined conditions is known as the proteome. Like the transcriptome, the proteome of an organism will also vary with external factors and conditions. 2 3 4 5

4. Master Layout (Part 1) 1 This animation consists of 3 parts: Part 1 – Genomic DNA contains large stretches of non-coding regions Part 2 – Single gene, multiple proteins Part 3 – Post-translational modification of proteins Genomic DNA 2 Transcription 5’ 5’ 3’ 3’ Exon 1 Intron Exon 2 Pre-mRNA 3 Spliceosome assembly 4 Lariat Exon 1 Exon 2 5 Mature mRNA Biochemistry by Stryer et al., 5th edition (ebook)

5. Definitions of the components:Part 1 – Genomic DNA contains large stretches of non-coding regions 1 1. Genomic DNA: The deoxyribonucleic acid polymeric sequence that acts as the store for genetic information and is essential for the synthesis of RNA and protein molecules, which are necessary for cellular functioning in all organisms. 2. Transcription: The process by which the genomic DNA is converted into a chemically related molecule, the messenger RNA or mRNA. Several enzymes and other factors are involved in this process. All regions of the DNA, coding and non-coding, get transcribed into the corresponding mRNA. 3. Pre-mRNA: The mRNA transcript that is produced from DNA as soon as transcription ends is known as the pre-mRNA. This contains both coding and non-coding sequences, is short lived and is further processed before translation. 4. Exon: The regions of the mRNA that code for specific portions or entire protein products upon translation are known as exons. They are often discontinuous with intervening nucleic acid sequences being present between them. 5. Intron: The intragenic sequences, sometimes considered as “junk”, in the pre-mRNA that do not get translated into proteins are known as introns. These are removed during the process of RNA splicing. 2 3 4 5

6. Definitions of the components:Part 1 – Genomic DNA contains large stretches of non-coding regions 1 6. Spliceosome assembly: This is made up of several protein-RNA complexes that bind the pre-mRNA and carry out splicing of the introns to give the mature mRNA. They are also commonly known as “snRNPs” or small nuclear ribonucleoproteins. These recognise specific sequences within the intron where they bind. 7. Lariat: The intron sequence that is removed by the spliceosome assembly forms a loop like structure with its 5’ end bound to an internal branch-point adenine residue. This is referred to as the lariat. 8. Mature mRNA: The mRNA that consists of only those nucleic acid sequences that are translated into proteins is known as the mature mRNA. 2 3 4 5

7. Part 1, Step 1: 1 RNAP Transcription 5’ 5’ 3’ 3’ Genomic DNA 3’ 3’ 5’ 5’ Template strand 2 Transcription factors Gene 5’ 3 RNAP 3’ Pre- mRNA released 3’ 3’ mRNA transcript 5’ 5’ RNAP dissociates RNAP 4 Action Description of the action Audio Narration Pre-mRNA is synthesized from genomic DNA by the process of transcription. The gene to be transcribed is bound by transcription initiation factors and then by RNA Polymerase which transcribes the gene in the 5’ to 3’ direction. The DNA strand that gets transcribed is known as the template strand. Once the termination sequences are reached, the enzyme and the newly formed mRNA transcript are released. First show the two blue strands on top followed by the colored circles appearing on the left of the strands. Next the red oval labelled ‘RNAP’ must bind to the blue strands. The strands must separate as shown in the middle panel and the RNAP oval must move across it. As it moves, the green strand shown must continuously appear behind it. This must continue until the RNAP reaches the end. Here it must dissociate from the blue strands along with the completed green strand as depicted. As shown in animation. 5

8. Part 1, Step 2: 1 Spliceosome assembly Intron A Exon 2 GU AG 2 Exon 1 Pre-mRNA Recognition elements for spliceosome assembly 5’ 3’ 3 5’ Conformation change 3’ 4 Action Description of the action Audio Narration The chain should change conformation as shown after binding of the red and purple shapes. The genomic DNA that gets transcribed into mRNA contains both exons, the coding sequences, as well as introns which are intervening, non-coding sequences . This pre-mRNA has certain recognition sites within its intron sequence that allows the spliceosome assembly to recognize it and bind to it. There is a conformation change that takes place upon binding of the protein-RNA complex. (Redraw all figures shown) First show the coloured intertwined chain on top followed by the arrow.Then show the tricolour chain followed by the alphabets and its label. Next, show binding of the red and purple shapes at the site shown. Once bound, the shape of the chain must change as shown below. 5 Biochemistry by Stryer et al., 5th edition (ebook)

9. Part 1, Step 3: 1 Binding of all snRNPs 2 Cleavage at 5’ end of intron Exon 1 with free 3’ end 3 5’ Lariat 5’ 3’ 3’ 4 Action Description of the action Audio Narration The remaining snRNPs bind following the conformation change of the pre-mRNA and there is cleavage at the GU site on the 5’ end of the intron. This then attaches to the branch site adenine nucleotide near its 3’ end to form the lariat structure. The pink and light blue circles must then bind followed by cleavage at the site indicated. The pink and light blue circles must also be shown to bind to the assembly. Next, there must be a break occurring at the site indicated and the green rod must be shown free at that end. 5

10. Part 1, Step 4: 1 Binding of 3’ end of exon 1 to 5’ end of exon 2 Unlike DNA, Proteins do not contain intervening sequences and are directly indicative of cellular function. 5’ 2 Cleavage at 3’ end of intron 3’ 3 Degraded 5’ Exon 2 Exon 1 3’ Mature mRNA 4 Action Description of the action Audio Narration The assembly then cleaves the 3’ end of the intron sequence containing the AG recognition element. The free 3’ hydroxyl group of the first exon attacks the 5’ end of the second exon such that they are joined to give the mature mRNA. The blue circular structure must get detached at the other end also as shown and the green and purple ends must join. First show the blue circle being broken at the other end also as shown. The green rod then binds the purple rod as indicated by the arrow mark. The blue circle along with the other group of shapes is removed as shown. 5

11. Master Layout (Part 2) 1 This animation consists of 3 parts: Part 1 – Genomic DNA contains large stretches of non-coding regions Part 2 – Single gene, multiple proteins Part 3 – Post-translational modification of proteins Pre-mRNA 2 Exon 1 Intron Intron Intron Exon 2 Exon 3 Exon 4 Alternative splicing Exon 3 Exon 4 Exon 1 Exon 1 Exon 2 Exon 4 3 Mature mRNA-A Mature mRNA-B Translation 4 Protein A Protein B 5

12. Definitions of the components:Part 2 – single gene, multiple proteins 1 1. Alternative splicing: This is a process by which the exons or coding sequences of the pre-mRNA produced by transcription of a gene are combined in different ways during RNA splicing. The resulting mature mRNA give rise to different protein products by translation, most of which are isoforms of one another. In this way a single gene can give rise to multiple protein products. 2. Translation:A process by which the mRNA sequence is read in the form of three letter codes known as codons to incorporate the corresponding amino acids in the growing polypeptide chain with the active involvement of rRNA, tRNA and several other enzymes. 3. Proteins A, B: The protein products resulting from translation of the differently spliced mRNA. Mature mRNA with exons 1, 2 and 4 gives rise to protein A while mRNA with exons 1,3 and 4 gives protein B. 2 3 4 5

13. Part 2, Step 1: 1 Pre-mRNA Exon 1 Intron Intron Intron Exon 2 Exon 3 Exon 4 2 Spliceosome assembly Alternative splicing Exon 1 Exon 3 Exon 4 Exon 2 Exon 4 Exon 1 3 Mature mRNA-A Mature mRNA-B 4 Action Description of the action Audio Narration First show the multicolored chain shown above with its various label markings. Next show the colored circular shapes appearing after the red ‘exon 1’. This assembly must act like a pair of scissors and first cut the red, blue and green parts of the chain out, which must rejoin below to give the ‘mature mRNA-A’. Next, from another intact chain, the red, violet and green parts of the chain must be cut out and these must rejoin to form the ‘mature mRNA-B’ shown on the right. Pre-mRNA transcribed from genomic DNA is often made up of several coding exons interspersed by non-coding introns. Alternative splicing, a common phenomenon observed in eukaryotes, allows the exons to be reconnected in multiple ways. There are several mechanisms for alternative splicing, the most common being exon skipping, wherein a particular exon may be included in the mature mRNA under specific conditions or in certain tissues and omitted from others. The multicolored chain shown above must be combined in different ways to give rise to the figures below. 5

14. Part 2, Step 2: 1 Translation Amino acid Ribosome tRNA Mature mRNA 3’ 2 3’ 5’ Movement of ribosome 5’ Growing polypeptide chain 3 3’ 3’ 5’ 5’ 4 Action Description of the action Audio Narration First show the figure on top left. Next show the blue and orange rect+circle coming in at position shown in fig 2 along with the other 3 rect+circles coming in slowly from the side. Next, the blue circle must be transferred on top of orange and blue rect must leave as shown in fig.3. The pink units must then move towards the right such the orange rectangle is now in same place where the blue was earlier. Green rectangle+circle must enter in position of orange and remaining 3 must slowly come in. This displacement must continue to happen as shown in fig.4 with the circles being continuously added on top of each other. The pink ‘ribosome’ must move along the blue strand with the rectangle+circle coming in one at a time. The mature mRNA produced then undergoes translation where it is bound to the ribosome and read as three letter codons. The corresponding amino acids are incorporated with the help of tRNAs. The ribosome moves along the mRNA and continues to incorporate the amino acids to the growing polypeptide until the termination codon is reached. 5

15. Part 2, Step 3: 1 Translation Mature mRNA-A Protein A 2 Translation Exon 1 Exon 3 Exon 4 Exon 2 Exon 4 Exon 1 3 Protein B Mature mRNA-B The proteome is significantly more complex than the genome since a single gene can encode multiple proteins. Without studying the proteome, it will not be possible to understand the conditions under which a particular protein is expressed. 4 Action Description of the action Audio Narration The colored chains must be shown to give to different ‘proteins’. The diversity of proteins encoded by a genome is greatly increased due to alternative splicing. Each mature mRNA formed gives rise to different protein products upon translation. Complexity of the proteome is therefore understood from the fact the a single gene can code for multiple proteins. First show the first colored chain giving rise to the blue ‘protein A’ followed by the second colored chain giving ‘protein B’. This must be followed by appearance of the textbox below. 5

16. Master Layout (Part 3) 1 This animation consists of 3 parts: Part 1 – Genomic DNA contains large stretches of non-coding regions Part 2 – Single gene, multiple proteins Part 3 – Post-translational modification of proteins 2 Post-translational modification Protein Hydroxylation Phosphorylation Methylation Glycosylation 3 CH3 CH3 CH3 CH3 CH3 OH OH OH OH Glucose Glucose Glucose Glucose P P P P P 4 Modified proteins 5

17. Definitions of the components:Part 3 – Post-translational modification of proteins 1 1. Protein: The polypeptide chain made up of several amino acid residues gets released at the end of the translation process and undergoes appropriate folding into its secondary and tertiary structures. If the protein is made up of multiple subunits, these come together to form the native protein structure. 2. Post-translational modification (PTM): Many proteins undergo chemical modifications at some of their amino acid residues after translation. These are carried out by enzyme catalyzed reactions and are essential for normal functioning of the protein. Some of the most commonly observed PTMs include: a) Phosphorylation: The addition of a phosphate group, usually to serine, threonine, tyrosine or histidine residues of the protein. Protein phosphorylation and dephosphorylation is one of the most important control mechanisms for inter-conversion of proteins between their functional and non-functional states. b) Glycosylation: The enzymatic addition of saccharides to specific amino acid residues resulting in the formation of glycoproteins. Sugars like glucose and mannose are commonly added to either nitrogen atoms of aspargine, arginine or to hydroxyl oxygen atoms of serine, threonine, tyrosine etc. c) Methylation: Addition of a methyl group, usually at lysine or arginine residues. d) Hydroxylation: Addition of a hydroxyl (-OH) group by the hydroxylase enzymes. Proline is usually the principal residue that is hydroxylated resulting in hydroxyproline, an essential and abundant component of connective tissues like collagen. 3. Modified protein: The protein that has undergone the required PTMs and is ready to function is the modified protein in its native, stable state conformation. 2 3 4 5

18. Part 3, Step 1: 1 The final functional protein structure most often does not correlate directly with the genome sequence due to several PTMs made in the protein. This further increases complexity of the proteome. Post-translational modifications 2 Protein Hydroxylation Phosphorylation Methylation Glycosylation 3 CH3 CH3 CH3 CH3 CH3 OH OH OH OH Glucose Glucose Glucose Glucose P P P P P Modified proteins 4 Action Description of the action Audio Narration The protein obtained by translation undergoes folding and various PTMs such as phosphorylation, alkylation, glycosylation, hydroxylation etc. to give the final functional protein. This adds to the complexity of each protein since the functional protein product does not directly correspond to its gene sequence. Please redraw figure. First show the blue chain on top. Then show the various groups shown in the figures below being added to the blue chain and appearance of the images below as shown followed by the text box above. The blue chain on top must be shown to which the groups shown in figures below must be added. 5

19. Interactivity option 1:Step No:1 1 Vs 4. Gives the true picture of functioning of an organism. 1. Expression levels are directly related to cellular activity. PROTEOMICS GENOMICS 2 5. Undergoes no further modifications once in its functional state. 2. Can give rise to multiple products which need to be further determined through experiments. 3 6. May not correspond to the final product which could be modified. 3. Contains large intervening sequences. 4 Results Interacativity Type Options Boundary/limits Once the user drags and drops a box under one column, it must turn green if correct or red if wrong. Box 1, 4 and 5 must come under ‘Proteomics’ column while Box 2, 3 and 6 must come under ‘Genomics’ column. User must be allowed to drag the boxes shown above and drop them under the correct column. Drag and drop 5

20. Questionnaire 1 1. A pre-mRNA sample has 150 nucleotides with two introns and two exons. Both the introns are 30 nucleotides long. What will be the length of the final mature mRNA? Answers: a) 100 b) 85 c) 90 d)‏ 150 2. Which enzyme is responsible for the process of transcription? Answers: a) DNA Polymerase b) RNA Polymerase c) Peptidyltransferase d)‏AminoacyltRNAsynthetase 3. The process of translation does not require which of the following? Answers: a) mRNA b) Ribosome c) tRNAd)‏ DNA 4. The lariat structure is formed during which of the following processes? Answers: a) Replication b) Translation c) Transcription d)‏ Splicing 2 3 4 5

21. Links for further reading Books: Discovering Genomics, Proteomics & Bioinformatics (2nd edition), A. Malcolm Campbell & Laurie J. Heyer Research papers: • Pandey, A. & Mann, M. Proteomics to study genes and genomics. Nature 2000, 405, 837-846. • Phizicky, E., Bastiaens, P. I. H., Zhu, H., Snyder, M., Fields, S., Protein analysis on a proteomic scale. Nature 2003, 422,208–215. • Wilkins, M. R., Williams, K. L., Apple, R. D. & Hochstrasser, D. F. Proteome Research: New Frontiers in Functional Genomics 1–243 (Springer, Berlin, 1997).