Transcription and Translation

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Transcription and Translation :

Transcription and Translation Prepared & Presented by Dr. Siva Reddy Challa, Professor & HOD, Dept. of Pharmacology KVSR Siddhartha College of Pharmaceutical Sciences, Siddhartha Nagar, Vijayawada-520010 Andhra Pradesh, INDIA Email: sivareddypharma@gmail.com

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Why should a cell carry out transcription and translation? Genetic codes in the DNA(A,T,G,C) transferred into RNA expressed into Proteins. Carry out the various activities inside a living cell eg.

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mRNA tRNA rRNA snRNA RNA transcript processing RNA transcript Transcription splicesome Ribosomal subunits tRNA tRNA-a.a. Translation by ribosome DNA transcription translation Nucleus cytoplasm

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Transcription Initiation: ATATTT TATAAA Pol II RNA Pol II and other transcription factor (TF) join in to form the holoenzyme that initiates transcription. ATATTT TATAAA Basal transcription factors binds to the TATA box of the promoter to form the basal transcription initiation complex. Transcription start site Promoter region

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Pol II unwinds DNA duplex, forming locally unwound segment of DNA (localized separation of the DNA helix). transcription bubble holoenzyme RNA chain is being synthesized by the Pol II holoenzyme, complementary to the template strand. 5’ 3’ 5’ 3’ template strand 5’

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5’ end of RNA modified by the ddition of 7-methyl guanosine, called the 5’ cap 5’ 3’ 5’ 3’ 5’ cap Elongation 5’ 3’ 5’ 3’ Pol II unwinds DNA helix ahead and rewind DNA strands behind the polymerization sites while moving along the DNA helix. More ribonucleotides added, and RNA chain is elongated. 5’

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Termination 5’ 3’ 5’ 3’ 5’ cap AAUAAA After the conserved sequence AAUAAA is being synthesized, the RNA transcript is cleaved by an endonuclease (endonucleolytic cleavage) 5’ cap AAUAAA 3’ endonucleolytic cleavage

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RNA transcript processing Addition of 7-Methyl guanosine (the 5’ cap) to the 5’end of an elongating RNA transcript. For: -- recognition by protein factor involved in the initiation of translation. -- protecting the growing RNA chain from nucleases. 5’ cap AAUAAA (2) Polyadenylation after the endonucleolytic cleavage of the RNA transcript. About 200 adenosine monophosphate residues added, forming the poly(A)tail. 3’ AAA………AAA Poly(A) tail

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(3) RNA splicing: removal of intron sequences from the primary RNA transcript. RNA splicing mRNA (exon sequence only) Primary RNA transcript (both intron & exon sequence) transcription exon 1 intron 1 exon 2 intron 2 exon 3 Gene (DNA)

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Comparing Replication (DNA synthesis) And Transcription (RNA synthesis) Transcription is very similar to replication except: The precursors are ribonucleotides instead of deoxyribonucleotides. RNA chains can be initiated de novo , does not require primase to synthesize the primer strand. Transcription uses one strand of DNA as template but replication uses both strands of DNA as template. Transcription uses RNA Polymerase while replication uses DNA Polymerase.

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Only one strand of DNA is used as a template 5’-CGTATGCTAGTCCGATTGCG-3’ 3’-GCATACGATCAGGCTAACGC-5’ 5’ 5’ 3’ 3’ Template strand Nontemplate strand 5’-CGUAUGCUAGUCCGAUUGCG-3’ mRNA 3’ 5’ transcription

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In Prokaryotes: Transcription and translation are coupled, ie. they occur simultaneously, prokaryotes Prokaryotes has no nuclear envelope, protein synthesis machinery can access the RNA. Eukaryotes: has nuclear envelope, RNA has to be transported out into the cytoplasm for protein synthesis. Why? but this does not occur in eukaryotes.

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translation Synthesis of protein. Components: Ribosomes mRNA aminoacyl-tRNA provide the macromolecular components required Cells devote more energy to protein synthesis than any other processes in the cell.

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Composition of ribosomes: 49 ribosomal proteins + 5S rRNA + 5.8S rRNA + 28S rRNA 33 ribosomal proteins + 18S rRNA 80S ribosome 60S subunit 40S subunit

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Components: Ribosomes mRNA aminoacyl-tRNA provide the macromolecular components required provide specifications for the amino acid sequence of the polypeptide. as adaptor molecule that incorporates amino acids into the polypeptides in response to the codons in the mRNA

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UAC a.a UCG a.a AAG a.a Aminoacyl-tRNA: anticodons mRNA: 5’-AGCCGCGGAUGUUCAGCAUAGCG……UAAGACCAAU-3’ 5’ untranslated region 3’ untranslated region codons start stop

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t-RNA anticodon Amino acid attachment

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Translation: (a) initiation (b) elongation (c termination ) Initiation: Cap-binding protein (CBP) binds to the 5’ cap at the 5’ end of mRNA, follow by binding of other initiation factors, and the small (40S) subunit of the ribosome.

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E P A Met 5’ 3’ The initiator methionyl-tRNA enters the P site The entire initiation complex moves 5’ to 3’ along the mRNA, searching for the AUG start codon. E P A Met 5’ 3’

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At the AUG codon, initiation factors dissociate, the large (60S) subunit binds to form the complete (80S) ribosome. E P A Met 5’ 3’ Another aminoacyl-tRNA enters the A site, dictacted by the codon sequence of the mRNA in the A site. E P A Met a.a.

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Transfer of the growing polypeptide chain from the tRNA at P site to the tRNA at A site by the formation of a new peptide bond, catalyzed by peptidyl transferase. E P A Met a.a. Elongation Peptide bond Translocation of ribosome to position the next codon at the A site. The nascent polypeptide-tRNA & the uncharged tRNA translocated from the A and P site to P and E site respectively. E P A a.a. Met

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Another aminoacyl-tRNA enters A site. a.a. E P A a.a. Met E P A a.a. Met a.a.

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E P A a.a. Met a.a. Transfer of the growing polypeptide chain from the tRNA at P site to the tRNA at A site by the formation of a new peptide bond. Uncharged tRNA exit E site. Repeat……. Translocation of ribosome to position the next codon at the A site. E P A a.a. Met a.a.

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termination : occurs when any of the termination codons UAA, UAG or UGA enters the A site E P A a.a. a.a a.a. a.a Met Release factor enters A site, uncharged tRNA leaves E site. Ribosome positions the A site at the termination codon. E P A a.a. a.a a.a. a.a Met

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All the components dissociate. E P A Polypeptide & release factor are released from the ribosome. Uncharged tRNA enters E site. a.a. a.a a.a. a.a Met

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The genetic codes Each genetic code consists of nucleotide triplets: 3 nucleotides in mRNA (codon) specify one amino acid. Genetic code is nonoverlapping: each nucleotide in mRNA belongs to one codon. ---AGGGCACCCUUAUCGCAAUGGGCUUAA--- codons

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Genetic code is ordered: multiple codons for a single amino acid, and codons for a.a. which have similar chemical properties are usually only differ by one nucleotide. Genetic code is degenerate: all but two of the amino acids are specified by more than one codon. UUU UUC GAU GAC Phe Asp GAA GAG Glu Similar chemical properties

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The genetic codes: Start codon: AUG Stop codon: UAA UAG UGA

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Tetracycline blocks binding of tRNA to A-site Streptomycin prevent transition from initiation to elongation step Chloramphenicol blocks peptidyl transferase reaction Rifamycin prevent initiation of RNA chains targets prokaryotes only, not on eukaryotes Inhibitors of transcription and translation as antibiotics Medical perspective:

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Regulation of Gene expression Transcriptional level Processing level Translational level Transcriptional level -determine whether a particular gene can be transcribed, if so, how often. -by group of proteins called transcription factor (TF), via protein-DNA interaction. eg. 1: special TF binds to enhancer sequence to control transcription of a particular gene.

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eg. 1: regulation of gene expression by steroid hormone (extracellular stimuli) Steroid hormone enters cell, combines with a receptor protein Hormone-receptor complex binds to a hormone response element in the DNA The bound complex stimulates transcription Transcript is processed and transported to the cytoplasm mRNA is translated into proteins

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eg. 2: Regulation of gene expression by peptide hormone (extracellular stimuli). Hormone binds to receptor protein in the membrane Hormone-receptor complex activates a cytoplasmic protein A signal is transduced to the nucleus Signal induces a transcription factor to bind to DNA Transcription factor stimulates transcription Transcript is processed and transported to the cytoplasm mRNA translated into proteins

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-affected by chromosome structure, eg. in euchromatin and heterochromatin. Why? (2) Processing level -eukaryotic genes--multiple introns and exons--- alternate splicing produces different mRNA, therefore different protein. (3) Translational level -localization of the mRNA -control of mRNA stability, the rate of mRNA degradation. -control of mRNA translation, when to translate? -assembly of ribosomes on the mRNA.

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Regulation of gene expression Transcriptional level Processing level Translational level Regulation of protein concentration Transcriptional level Processing level Translational level Posttranslational level (protein degradation)

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Posttranslational control: controlling protein stability DNA mRNA protein degraded Proteasome : protein-degrading machine. Digest proteins that have been specifically marked by the ubiquitin molecule (a small protein molecule). * * * *

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Points about transcription Transcription = synthesis of RNA using DNA as template. Catalyzed by RNA polymerase that adds ribonucleotide to the 3’-OH of the RNA chain. RNA polymerase functions in the form of a complex with many other proteins — collectively known as the holoenzyme . 3 RNA polymerases: RNA polymerase I, II, III --- each transcribe different sets of genes. RNA Pol II for mRNA.

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3 stages: initiation, elongation and termination The RNA is complementary to one of the strands of DNA the template strand. mRNA (messenger RNA) is the messenger that transfer the genetic information from the nucleus to the cytoplasm. Initiation: RNA pol with the help of proteins called transcription factors (TF), binds to the promoter region of the gene.

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Elongation: RNA Pol moves from the 3’ end of the template strand towards 5’ end, thus, RNA chain is growing in the 5’ to 3’ direction. Ribonucleotides (A,G,C,U) are being added according to the Watson-Crick base paring. eg. DNA: --GCTA-- RNA: --CGAU-- Transcription occurs within a locally unwound segment of DNA --- the transcription bubble. The unwound helix provides the single-stranded template strand to base-pair with the incoming ribonucleotides.

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RNA Pol can unwind the DNA helix in front of the polymerization site to allow base-paring to occur, and rewind the DNA strands back into helix behind the polymerization site. Termination: occurs after RNA Pol synthesized the sequence AAUAAA, RNA chain cleaved by endonuclease before the holoenzyme actually stops and dissociate from the DNA template.

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Terminology: template strand = transcribed strand = non-coding = Antisense strand non-template strand = non-transcribed strand = coding strand = Sense strand

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Points about translation Basic components required are the ribosome, mRNA and aminoacyl-tRNA. Eukaryotic ribosome is 80S, made up of the 60S large subunit and the 40S small subunit. Translation starts at the start codon AUG and stops at the stop codons UAA, UAG & UGA. Codons are the nucleotide triplet sequence on mRNA, anticodons are the nucleotide triplet sequence on tRNA, which form base paring with the codon .

Slide 42:

Translation initiation, elongation and termintion requires multiple initiation factors, elongation factors and termination factor. To initiate translation, only the initiator tRNA can enter the P site directly, all other a.a.-tRNA will enter the A site during elongation. During elongation, the growing polypeptide chain is transferred from the tRNA in P site to the aminoacyl-tRNA at A site. Peptide bond is formed by the peptidyl transferase.

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Ribosome translocate from the 5’ end of mRNA towards the 3’ end. The polypeptide is extending from the amino terminal (-NH 2 ) towards the carboxyl terminal (-COOH).

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Thank You