Gene expression

A clear compilation of various steps constituting gene expression in living organisms. Explained in simple words, pictorial representation and intriguing animations.A dive into the concepts of Transcription and translation and associated modifications that lead to protein synthesis in Eukaryotes and Prokaryotes. Science made Fun and easy. - Cellular & Molecular Biology.

1. GENE EXPRESSION PRESENTED BY: Ayesha nazeer I mpharm ( Dept. of pharmacology) 2020-2022 Srinivas college of pharmacy, mangalore .

2. CONTENTS INTRODUCTION ……………………………………………………………………………………………………….PG. NO. 3 TRANSCRIPTION………………………………………………………………………………………………………..PG. NO. 4 TRANSCRIPTION IN PROKARYOTES………………………………………………………………………………PG. NO. 5 - 9 TRANSCRIPTION IN EUKARYOTES…………………………………………………………………………………PG. NO. 10 - 17 TRANSLATION…………………………………………………………………………………………………............PG. NO. 18 - 30 POST-TRANSLATIONAL MODIFICATIONS: CHAPERONES …………………….………………………….PG. NO. 31 – 34 5) REFERENCE…………………………………………………………………………………………………………….PG. NO. 35 2

3. GENE EXPRESSION Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product(RNA) that enable to produce protein as the end product. It is controlled at various points in sequence leading to protein synthesis. STEPS: Transcription, Translation, Post translational modifications, Protein folding, Protein Transport. Transcriptome: set of all RNA molecules in one cell or a population of cells. Initial product of expression Proteome: Entire range of proteins & their biological function. Final product of Gene expression. 3

4. transcription Transcription is the first of several steps of DNA based gene expression in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Coding strand/ non-template strand (same N-bases as RNA formed) Template strand/ non-coding strand. (source to form RNA having complementary N-bases ) 4

5. TRANSCRIPTION IN PROKARYOTES DNA is transcribed by the enzyme RNA Polymerase . Where first 5 subunits form the core enzyme( catalytic activity). But along with sigma factor(promoter recognition) they form the Holoenzyme (Functionally active) CRUCIAL ACTIONS : Binding to DNA. Moves along the DNA, unwinding the helix at its active site Indicated by Mg2+ which is required for catalysis. It adds nucleotides to the RNA chain at polymerization site, using exposed DNA strand as template. Hence Forms complementary copy RNA. Short region of DNA/RNA helix is formed only transiently. 5

6. INITIATION ELONGATION TERMINATION RNA polymerase holoenzyme assembles( core-enzyme+ sigma factor) Locates PROMOTER DNA sequence. Polymerase unwinds the DNA at position at which transcription to begin( active site) RNA sequence starts to add. Once 10 nucleotide sequence formed. Breaks interaction with promoter DNA and releases sigma factor. Elongation begins and RNA tightens itself around DNA moving along the DNA. Newly transcribed RNA releases when termination signal is encountered. Termination signal is encoded in DNA. And also can be attained by the hair- pin like structure of RNA that destabilizes the polymerase’s hold on the RNA. INITIATION ELONGATION TERMINATION 6

7. INITIATION Promoter sites: Base sequences for recognition: Pribnow box: 6 nucleotide bases (TATAAT) Located on left, 10 bases away from origin of transcription. The 35’ sequence: 2 nd recognition site in promoter region Base sequence TTGACA Located 35 bases away on left from site of transcription. 7

8. ELONGATION Ribonucleotide triphosphate (ATP,GTP,CTP,UTP) used for RNA formation in 5’ 3’ direction. For each nucleotide addition a pyrophosphate released. Sequence is complementary to template, identical to coding (RNA-U, DNA- T) RNAP- no primer, no endo/exonuclease activity, no repair 8

9. TERMINATION Transcription stops by signals. Rho ( ρ ) dependent termination ρ factor- Binds to growing RNA and unwinds the DNA-RNA hybrid and RNAP can’t move further and dissociates from the DNA. ATPase- terminates the process, releases RNA Dissociation of RNA polymerase. Rho ( ρ ) independent termination. Palindrome like bases occur at the end sequence of the DNA. Due to these sequence the newly synthesized RNA folds on itself and leads to Formation of hairpins of RNA (loop formed due to complementary base pairing) This structure destabilizes the RNAP’s hold on the RNA. 9

10. TRANSCRITION IN EUKARYOTES RNA synthesis : complicated RNA polymerase: RNA polymerase I- synthesis of large rRNAs precursor RNA polymerase II- synthesis of mRNAs, snRNA(small nuclear RNA) precursor RNA polymerase III- formation of tRNAs & srRNA (small regulatory RNA) 10

11. To begin, RNA polymerase requires several general transcription factors.( humans contain 6 TFs) The promoter contains a DNA sequence called the TATA box , located 25 nucleotides away from site of initiation. B) Through its subunit TBP, TFIID first recognises and binds to the TATA Box .( hogness box ) C) which then enable adjacent binding of TFIIA and TFIIB .  The binding of TFIID to DNA produces a distortion due to the DNA bending by TBP(not shown in flow-chart)– this serves as a landmark to attract the other general transcriptional factors. D) The rest of the general transcriptional factors ( TFIIF, TFIIE & TFIIH ) and the RNA polymerase enzyme itself, assemble at the promoter. E) TFIIH uses the energy from ATP hydrolysis to pry apart the DNA helix at start point, exposing the template strand. In addition it phosphorylates RNAP II so that the enzyme is released from general factors and can begin the elongation phase. INITITATION 11

12. Promoter site: Sequence of DNA bases, almost identical to pribnow box(prokaryote) Known as Hogness bog(TATA box) Left (25 nucleotides from mRNA synthesis starting site) CAAT box - another site of recognition ,70-80 nucleotides upstream from start of transcription. One out of the above help RNAP II to re cognize the required DNA sequence or transcription. STIMULATION : Enhancers increase gene expression by about 100 folds. This is possible by binding of enhancers to the transcription factors to form activators . Activators also attract ATP-dependent chromatin remodelling complexes and histone modifying enzymes . 12

13. TERMINATION and polyadenylation 13

14. Post transcriptional modifications (in eukaryotes.) RNA polymerase II-----primary mRNA transcript ( Heterogenous nuclear RNA) Processed form-mRNA (protein synthesis) Includes- Terminal base modification( 5’ capping / Poly A tail) Removal of introns/ Joining of exons( RNA Splicing) Export of spliced RNA to cytosol. Post –transcriptional modifications of mRNA occurs in nucleus. The mature RNA then enters into the cytosol for translat ion. 1)5’ capping;- This process is the first of the processing reactions for hnRNA . The cap is 7-methyl guanosine attached ‘backward’ to the 5’ terminal end of mRNA forming 5’→5’ triphosphate linkage. Methylation of this terminal guanine is catalyzed by guanine -7-methyl transferase. 5-adenosyl methionine is the source of methyl group. Addition of 7-methyl guanosine permits initiation of translation & helps in stabilization of mRNA. 14

15. 2.) Poly-A tail addition mRNA- adenine nucleotide chain at 3’end Not during transcription, after termination by cleavage to stabilize mRNA Reduced while entering cytosol. (explained via chart in earlier slide) Removal of introns ;- Maturation of eukaryotic mRNA usually involves removal of RNA sequences which do not code for protein (introns) from primary transcript.. The remaining coding sequence , the exons are spliced together to form mature mRNA. Introns can be removed by the SnRNP(small nuclear Ribo nucleo protein particle). Spliceosome = hnRNA + snRNP (formed at exon-intron junction) Ends of two exons join together. 5’-capping 15

16. Alternate splicing Alternate patterns of hnRNA splicing result in different mRNA sequences which can produce different proteins. Alternative splicing results in mRNA heterogeneity. Faulty splicing can cause diseases;- Splicing of hnRNA has to be performed with precision to produce functional RNA. Faulty splicing may result in diseases. E.g ;- β -thalassemia. this is due to a mutation that results in a nucleotide change at an exon-intron junction.  Decreased Beta chain haemoglobin Transfer RNA -All– post transcriptional modification Trimming, converting bases, addition of CCA nucleotides (3’end) Ribosomal RNA- Preribosomal post transcription modification ribosomal RNA 16

17. Inhibitors of transcription: Antibiotics & toxins Actinomycin D- Streptomyces sp. DNA template strand Blocks movement of RNA polymerase Rifampin- Tuberculosis, leprosy. Β subunit of prokaryotic RNA polymerase-binds- inhibits action α- amantin - Amantia phalloides RNA polymerase II-binds Reverse transcription: Retroviruses- RNA Oncogenic RNA dependent DNA polymerase/reverse transcriptase 17

18. translation Biosynthesis of a protein/polypeptide Components required for protein synthesis: Amino acids ;- Proteins are polymers of amino acid 20 amino acids found in protein structure, 10  synthesized by humans. 10  provided through the diet-called as essential amino acids. deficiency in the dietary supply of any one of essential amino acid, the translation stops. Hence, regular dietary supply of essential amino acids should be maintained for proper protein synthesis. In prokaryotes , there is no requirement of dietary amino acid , since all the 20 are synthesized from the long inorganic components. Ribosomes ;- The ribosomes are factories for protein synthesis. Ribosomes are huge complex structures (70s for prokaryotes & 80s for eukaryotes) of proteins & ribosomal RNAs. Each ribosome consist of 2 subunits- one big & one small. 18

19. The functional ribosome has 2 sites- A site & P site . A site is for binding of aminoacyl tRNA & P site is for binding peptidyl tRNA . A site is acceptor site & P site is donor site. In case of eukaryotes, there is another site called exist site or E site Thus eukaryotes contains 3 sites (A,P & E). Messenger RNA- Information for protein synthesis. Transfer RNA- They carry the amino acid & hand them over the growing peptide chain. The amino acid is covalently bound to tRNA at 3’ end. Energy sources- Both ATP & GTP are required for supply of energy in protein synthesis. Protein factors- These are needed for initiation, elongation & termination. 19

20. CODONS: 3N base sequence- codons Total 4N bases exist- purines(A,G), pyrimidine(U,C) 64 combinations exist They are read From 5’ to 3’ 61 code for 20 amino acids UAA, UAG, UGA-don’t code-Nonsense/termination codons AUG- start codon characteristics of genetic code Universal: same codons, same aa. exceptions-AUA (methionine, mitochondria / isoleucine, cytoplasm) Specific: UGG-tryptophan Non-overlapping : Each nucleotide is part of only one codon and read only once. Continuous : no space or comma Degenerate : more than 1 codes for same aa, 61 codes 20aa. Synonyms, differ in 3 rd base 20

21. CODON-ANTICODON RECOGNITION: Codon in mRNA- recognized by anticodon of tRNA Pair-up in antiparallel direction Complementary pairing (A=U, C≡G) WOBBLE PAIRING The wobble base of the anticodon is the one at 5’ end, it forms H-bonds with the last base of mRNA at 3’ end. The third base of mRNA codon can tolerate mismatching more than the other two positions. 21

22. STEPS OF TRANSLATION 1.) ACTIVATION OF AMINO ACID BY SYNTHETASE ENZYME 2.) PROTEIN SYNTHESIS PROPER  INITIATION  ELONGATION  TERMINATION. 1.) ACTIVATION OF AMINO ACID BY SYNTHETASE ENZYME An amino acid is activated for protein synthesis by an amino- acyltRNA synthetase enzyme in 2 steps: (both steps require binding of the moieties to the enzyme) 1. First by linkage of aa’s carboxyl group to AMP  forms adenylated amino acid. (This AMP is obtained by hydrolysis of ATP). 2. the AMP-linkage of carboxyl grp of aa is broken first ,then the carboxyl grp is transferred to hydroxyl group on sugar at the 3’ end of tRNA. This joins the aa by an ‘activated’ ester linkage to tRNA and forms the amino acyl tRNA molecule( aka Activated Amino acid.) 22

23. 2. Protein synthesis proper: Protein synthesis by ribosome mRNA- read in 5’-3’ Polypeptide synthesis- from N to C end Prokaryotic mRNA-polycistronic- multiple polypeptide ( why?) Eukaryotic mRNA- monocistronic - single polypeptide 23

24. Initiation in EUKARYOTES- It is complex in eukaryotes. It involves at least 10 eukaryotic initiation factors( eIF ). Initiation process involves 4 stages Ribosomal dissociation Formation of 43s pre-initiation complex Formation of 48s initiation complex Formation of 80s initiation complex 1) ribosomal dissociation The 80s ribosome dissociate to form 40s & 60s subunits. 2 initiating factors eIF-3 & eIF-1/1 A ,bind to the newly formed 40s subunit & block its reassociation with 60s subunit. eIF-3 is called as anti-association factor. 2)formation of 43s pre initiation complex A ternary complex containing met-tRNA’ & eIF-2 bound to GTP attaches to 40s ribosomal subunit to form 43 pre initiation complex.  eIF-3 & eIF -1 A stabilizes this complex. 24

25. 3)formation of 48s initiation complex The binding of mRNA to 43s pre initiation complex results in the formation of 48s initiation complex. This involves interaction between some of eIFs & activation of mRNA. 48s initiation complex eIF-4F complex is formed by the association of eIF-4G with eIF-4E. eIF-4F formed binds to the cap of mRNA ,then eIF-4A and eIF-4B bind to mRNA & reduce its complex structure. 4)formation of 80s initiation complex 48s initiation complex binds to 60s ribosomal subunit to form 80s initiation complex This step is facilitated by involvement of eIF-5. 25

26. initiation in prokaryotes: It is less complex The 30s ribosomal subunit is bound to initiation factor 1 & 3 & attached to ternary complex ( IF-2,formyl met –tRNA & GTP). Shine- dalgarno sequence  8 bases upstream from AUG(start codon) this sequence helps to recruit the ribosome for translation to commence. The 50s ribosome unit is now bound with the 30s unit to produce 70s initiation complex in prokaryotes. Elongation of translation: (requires elongation factors) Binding of amino acyl tRNA to vacant A site of ribosome. Peptide bond formation. Translocation 26

27. Binding of amino acyl tRNA to A site 80s initiation complex has met-tRNA at its P site.(via AUG- start codon) A site is vacant. Aminoacyl tRNA(along with eEF1A+GTP) occupy this site. By Codon recognition on mRNA EF-1 A(in euk) / Ef-tu (in prok ) , GDP- recycled Peptide bond formation Peptidyl transferase forms peptide bond by condensation It was mistaken to be protein based enzyme but it is actually a RNA molecule with catalytic activity  aka RIBOZYME Growing peptide chain is transferred to tRNA of A site 27

28. TRANSLOCATION: The large subunit translocates relative to the small subunit unit first. Followed by translocation of small subunit along with mRNA. This resets the ribosome with fully empty A site, ready for next aminoacyl tRNA to bind. EF-G( in prok )/ EF-2(in euk) promotes ribosome translocation.(smaller subunit) Deacylated tRNA moves to E-site in eukaryotes. 28

29. TERMINATION : Stop codon terminates the growing polypeptide.( UAA, UAG and UGA) Ribosome encounters stop codon- there is no tRNA available to bind to A site of ribosome. Instead a release factor binds. Eukaryote release factor1( eRF1) recognizes all 3 stop codons. eRF3 stimulates termination events. Binding of release factors dismantles the ribosome unit. Small & large subunits are released. Polypeptide product is freed. Ribosome recycling  eukaryotes. eRF-GTP complex along with enzyme peptidyl transferase cleaves the peptide bond. 29

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31. POST TRANSLATIONAL MODIFICATIONS Not functional protein Protein folding , trimming by proteolytic degradation , intein splicing(catalyze own removal), covalent changes After removal of intein , precursor protein becomes biologically active. Phosphorylation: serine, threonine, tyrosine by Protein kinase, phosphatase. Hydroxylation: Proline, lysine  collagen ( by hydroxylase) Glycosylation: attach carbohydrate---serine, threonine, asparagine  glycoprotein 31

32. CHAPERONES: Molecular chaperones recognize incorrect folding due to the exposure of hydrophobic surfaces in misfolded proteins. In correctly folded proteins these surfaces are deeply buried inside. Binding of exposed hydrophobic surfaces causes aggregate of proteins  diseased condition in human. Chaperones bind to these exposed surfaces and help the proteins re-fold. Many chaperones are called Heat shock proteins( hsp ) Because They are synthesised in dramatically large amt when cells are exposed to high temp. protein misfolding and diseases: Failure to fold properly- rapid degradation Cystic fibrosis- autosomal recessive disease Degraded CFTR (cystic fibrosis transmembrane regulatory protein  normally responsible for transportation of Chloride ions+water from cells( lungs,pancreas etc )trapped ions& wateras response thick & sticky mucus formed. 32

33. MAJOR MOLECULAR CHAPERONE FAMILIES: (each cell organelle has its own set of hsps belonging to these families) Hsp70 proteins family: Acts early in proteins, i.e. before the protein leaves ribosome. Each monomer of hsp70 binds to string of aa. Using an ATP.  By binding tightly to partially synthesized peptide sequences (incomplete proteins), Hsp70 prevents them from aggregating and being rendered non-functional. ATP hydrolysis causes hsp70 to release the protein  protein free to fold on its own. Hsp60 proteins family: They form large barrel shaped structure. They act only after protein has been fully synthesized. Aka CHAPERONIN. Forms “isolation chambers” that captures the misfolded protein via hydrophobic entrance. Interior is lined with hydrophilic surface, sealed with a lid by utilising ATP. Protein is allowed to fold completely in isolation. Hydrolysis of that ATP pops open the lid. The protein is then released from chamber. 33

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35. REFERENCES DAVID FREIFELDER, MOLECULAR BIOLOGY, Second Edition, Page no. 307 - 356 35