Transcription: RNA Synthesis, Processing & Modification

Transcription

1 Transcription: RNA Synthesis, Processing & Modification 1

2 Central dogma DNA RNA Protein Reverse transcription 2

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 3

4 Transcription DNA 3 Template strand/antisense strandy RNA 5 DNA 3 4

5 3 Major kinds of RNA Messenger RNAs (mrnas) encode the a.a seq 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 ribosomescellular machines that synthesize proteins 5

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 6

7 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 RNA Polymerase Lack the proof reading active site more error 7

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

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 9

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 10

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 1 st nt is added start site/point 11

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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 13

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 14

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 15

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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 ligasessplicing process New exons cont seq that specifies a functional polypeptide 17

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. 18

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

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 20

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

22 TRANSLATION: PROTEIN SYNTHESIS AND GENE EXPRESSION 22

23 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 Introduction 23

24 Dictionary of Genetic Code 24

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) 25

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

27 Transfer RNA (TRNA) 27

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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 29

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

31 Activation of amino acid In cytosol, aminoacyl-trna synthetases esterify 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+t-RNA Aminoacyl AMP + Ppi (aminoacyl-adenylate complex) Aminoacyl Trna + Amp +Ppi 31

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-trna fmet trna met internal AUG - for initiation AUG Initiation in bacteria require: 70S rib, mrna, fmet-trna fmet 3 proteins initiation factors (IF-1, IF-2 and IF-3) 32

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 33

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 34

35 Initiation The initiating 5 AUG is positioned in at the P site- the only site fmettrna fmet can bind fmet-trna fmet is the only aminoacyl Trna 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 35

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

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 37

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 38

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 aminoacyl Trna-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 dissociatetime for codon anticodon interaction to be proofread. Incorrect aminoacyl-trnas normally dissociate from A site 39

40 Elongation Step 2: Peptide bond formation The peptidyl transferase catalyzing the formation of peptide bond by transferring N- formylmethionyl group to the amino group of the second amino acyl-trna in the A site forming dipeptidyl-trna The uncharged (deacylated) TRNA fmet remains bound to the P site 40

41 Elongation Step 3: Translocation The ribo moves one codon towards the 3 end of the MRNA This movement shifts the anticodon of the dipeptidyl Trna (which still attached to the 2 nd codon from A to P site At the same time the deacylated Trna 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 2 nd codon 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 41

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