Transcription: RNA Synthesis, Processing & Modification

1. Transcription:RNA Synthesis, Processing & Modification

2. Central dogma DNA → RNA → Protein Reverse transcription

3. Transcription • The process of making RNA from DNA • Produces all type of RNA –mRNA, tRNA, rRNA, snRNA, miRNA and siRNA • Ribont is produced rather than deoxyribont • U replaces T • A primer is not needed, but a DNA template is needed • Only a very small portion of the genome is transcribed or copied into RNA – entire genome must be copied during DNA replication • RNA chain from 5’ to 3’end • No proofreading

4. Transcription DNA 3’ Template strand/antisense strandy6 5’ RNA 5’ 3’ 5’ DNA 3’

5. 3 Major kinds of RNA • Messenger RNAs (mRNAs) – encode the a.aseq of one or more polypeptide specified by a gene or set of genes • Transfer RNAs(Trna) – read the information encoded in the Mrna and transfer the appropriate aa to a growing polypeptide chain during protein synthesis • Ribosomal RNAs-constituents of ribosomes-cellular machines that synthesize proteins

6. During replication – the entire chromosome is usually copied • Transcription is more selective • Only particular genes or groups of genes are transcribed at any one time – some portions of the DNA genome are never transcribed • Specific regulatory sequences mark the beginning and end of DNA segments to be transcribed and designate which strand of duplex DNA to be used as the template

7. RNA Polymerase • Synthesized the transcription • The most studied- in E.Coli • 5 different subunits –α2ωββ’σ (holoenzyme) • α2ωββ’– core enzyme • σ – recognize specific promoter (a DNA sequence that signals the start of RNA transcription) • α2ωββ‘ – make the active site for polymerization • Only holoenzyme can initiate transcription • Lack the proof reading active site – more error

8. Stages of transcription • Formation of transcription complex (of DNA and RNA polymerase) • Initiation • Elongation • Termination

9. Initiation-RNA synthesis begins at promoters • RNA Pol need to bind to specific sequence of DNA to start transcription - forms closed complex • These sequence – promoter • Sigma factor recognizes the promoter sequence • Mutation in promoter affect the efficiency of RNAP binding and transcription initiation

10. Characteristics of Promoter sequence • Pribnow box- sequence contained in the promoter region (5-10 bases to the left – upstream first four bases to be transcribed to RNA) • All Pribnow box found in eukaryotes are variant of TATAATG sequences – TATA BOX

11. Initiation-RNA synthesis begins at promoters • RNA Pol attaches to promoter region-forms a close complex, promoter DNA is stably bound but not unwound • RNA Pol melts the helical structure (~12-15bp from -10 region to +2 and +3) and separates the 2 strands of DNA locally – open promoter complex • RNA Pol initiates RNA synthesis. The site at which the 1st nt is added – start site/point

13. Chain elongation • After the strands have separated, a transcription bubble of about 17 bp moves down the DNA sequence to be transcribed • RNA Pol catalyzes the formation of the phosphodiester bonds between the incorporated ribont • About 10 nt is added, sigma s/u dissociates and is later recycled to bind to another RNA Pol core enzyme • The DNA helix reclosed after RNA Pol transcribes through it and growing RNA chain dissociates from DNA

14. Chain termination 1) Intrinsic termination/rho independent termination • Controlled by termination sites – specific sequences on the DNA molecule function as the signal for termination of transcription process • Two inverted repeats spaced by few other based followed by repeats of Adenosine • Inverted repeats – sequences of bases that are complementary, they can loop back on themselves • When the RNA is created, the inverted repeats form a hairpin loop and stall the advancement of RNA Pol • The presence of uracils cause a series of A-U base pairs between the template strand and the RNA, and relatively unstable • RNA dissociate from the transcription bubble- end of transcription

15. Chain termination 2) The rho (ρ) factor mechanism • Rho protein binds to the RNA and chases the RNA Pol. • When the RNA Pol pause at the termination site, the rho protein has a chance to catch up the RNA Pol • Rho proteins reaches the termination site, it facilitate the dissociation of the transcription machinery by unwinds the DNA-RNA hybrid in the transcription bubble

17. RNA Processing Additional modification in RNA after transcription 1)Splicing • Usually in eukaryotes • Primary transcript of Mrna contain of intron (non-coding region and exon (coding region) • Removal of intron by nucleases and joining of exons by ligases- splicing process • New exons – cont seq that specifies a functional polypeptide

18. RNA Processing 2) 5’ Cap • Usually in eukaryote • 7-methylguanosine linked to the 5’ terminal residue • 5’ cap helps protect Mrna from ribonucleases • Also binds to a specific complex of proteins and participates in binding of Mrna to the ribosome to initiate translation • Occur very early in transcription, after the first 20/30 nts are added.

19. RNA Processing 2) 3’ Poly A tail • Usually in eukaryote • 80-250 A residue is added to the 3’end (Poly A tail) • helps protect Mrna from ribonucleases

20. RNA degradation • Conc of any molecule depends on rate of synthesis and rate of degradation • Synth and degradation of an Mrna is balanced – a change in the process lead to accumulation or depletion • Degradative pathways ensure mrna do not build up in the cell and direct the synthesis of unnecessary proteins • Degradation depends on the need of the cell • If needed very briefly-half life of mrna maybe minutes/seconds • If needed constantly by the cell-can stable for many cell generation • Average in vertebrate – 3hours • Average in bacteria – 1.5min • Degradation by ribonucleases

21. TASK List down the antibiotics that inhibit the process of transcription and explain the mechanism of its action towards inhibiting the pathogen invasion

22. TRANSLATION:PROTEIN SYNTHESIS AND GENE EXPRESSION

23. Introduction • Protein are end products of most information pathways • A normal cell need thousand of different proteins at any given moment • They must be synthesized in response to the cell’s current needs, transported to their appropriate cellular locations and degraded when no longer needed • Protein synthesis is a complex process but still are made at exceedingly high rates • Polyp of 100 res is synth in E.Coli cell in only 5 sec • 2 key components in protein synth; ribosome and Trna

24. Dictionary of Genetic Code

25. Genetic code Important features: • Triplet • Non overlapping • Commaless- arranged as continuous structure • Degenerate – dissimilar components can perform a similar fx: UAU and UAC represent tyr • Universal code • There are 64 combinations of 3 bases producing 64 codons • Codons- triplet of nts that codes for a specific aa • Special codons: AUG (meth – start codon) (UAA, UAG, UGA – stop codon)

26. Ribosomes • E.coli contain >15000 ribosome • Bact ribosome: 65% Rrna and 35% proteins • Bact rib: 70S (50S+30S) • Euk rib: 80S (60+40S)

27. Transfer RNA (TRNA)

29. Translation of Mrna • A process to synthesize a protein from mRNA • The amino acid (aa) is added sequentially in a specific number and sequence, determined by the sequence of codons in the genetic code of the relevant mRNA

30. STEPS IN PROTEIN SYNTHESIS • Activation of amino acid • Initiation • Elongation • termination

31. Activation of amino acid • In cytosol, aminoacyl-Trnasynthetasesesterify the 20aa to their corresponding tRNA • Each enzyme specific for one aa • Formation of aminoacyl t-RNA • The amino acid need to be activated before they can be incorporated into the peptide chain • Attachment of the correct aa to the adaptor (fidelity) Amino acid + ATP Aminoacyl AMP + Ppi(aminoacyl-adenylate complex) Aminoacyl-AMP+t-RNA AminoacylTrna+ Amp +Ppi

32. Initiation • Prot synth begin at the amino (NH2) terminal and proceeds to the carboxyl (COO) terminal • AUG (methionin)- start codon • 2 types of RNA specific for Meth: • fmet-tRNAfmet- for initiation AUG • tRNAmet – internal AUG • Initiation in bacteria require: • 70S rib, mRNA, fmet-tRNAfmet • 3 proteins – initiation factors (IF-1, IF-2 and IF-3)

33. Initiation 1) Dissociation of ribosome • Before initiation process starts, 70S ribosome dissociate into 30S and 50S s/u • 2 initiation factor, IF-3 AND IF-1 binds to the newly dissociated 30S – To prevent re-association and allows other translation initiation factors to associate with 30S s/u and prepares it for formation of 70S initiation complex

34. Initiation • The association of ribosome and RNA will form preinitiation complex • Pre initiation complex is guided for initiation codon AUG by Shine Dalgarno sequence • Precise positioning is needed for initiation • Bacterial ribosomes have 3 sites: • Peptidyl (P) – binds a TRNA that carries a peptide chain • Aminoacyl (A) – binds incoming aminoacyl TRNA • E (exit)-carries uncharged TRNA that is about to be released from the ribosome

35. Initiation • The initiating 5’AUG is positioned in at the P site- the only site fMet-tRnafmetcan bind • fMet-tRnafmetis the only aminoacylTrna that binds first to the P site, as during the elongation stage all incoming aminoacyl-trna binds first to the A and only to the P and E • IF-1 binds at the A site and prevents Trna binding at this site during initiation

36. Initiation Step 2: • The complex is joined by both GTP bound IF-2 and fMet-tRnafmet • The anticodon of this Trna now pairs correctly with the Mrna’s initiation codon

37. Initiation Step 3: • This complex binds to 50S ribosomal su, and simultaneously the GTP bound to IF-2 is hydrolyzed to GDP and Pi and released from complex • All 3 IF depart from rib at this point • Completion of these steps produces a functional 70S rib – initiation complex • Now ready for elongation

38. Elongation Require: • The initiation complex • Aminoacyl-trnas • Elongation factors (EF-Tu, EF-Ts and EF-G) • GTP • Involve 3 steps and cells use these 3 steps to add aa residue and are repeated as many times as needed

39. Elongation Step 1: • Binding of an incoming Aminoacyl-Trna • The appropriate incoming aminoacyl-TRNA binds to a complex of GTP bound EF-TU – producing aminoacylTrna-EF-TU-GTP complex binds to the A site of the 70S initiation complex • EF-TU-GTP and EF-TU-GDP complexes exist for few ms b4 they dissociate- time for codonanticodon interaction to be proofread. • Incorrect aminoacyl-Trnas normally dissociate from A site

40. Elongation Step 2: Peptide bond formation • The peptidyltransferasecatalyzing the formation of peptide bond by transferring N-formylmethionyl group to the amino group of the second amino acyl-TRNA in the A site – formingdipeptidyl-TRNA • The uncharged (deacylated) TRNA fmet remains bound to the P site

41. Elongation Step 3: Translocation • The ribo moves one codon towards the 3’end of the MRNA • This movement shifts the anticodon of the dipeptidylTrna (which still attached to the 2ndcodon from A to P site • At the same time the deacylatedTrna is shifted from P to E site, and Trna is releases into the cytosol • The third codon of the mrna now lies in the A site and the 2ndcodon in the P site • Movement of rib along Mrna require EF-G (Translocases) and the energy is provided by hydrolysis of another molecule of GTP • The rib is now ready for next addition of aa

42. TASK • List down the antibiotics that inhibit the process of translation and explain the mechanism of its action towards inhibiting the pathogen invasion