PROTEIN SYNTHESIS 1 PROTEIN SYNTHESIS q DNA NUCLEIC

PROTEIN SYNTHESIS 1

PROTEIN SYNTHESIS q DNA: NUCLEIC ACID, DOUBLE STRAND, PO 4, DEOXYRIBOSE SUGAR. q RNA: NUCLEIC ACID, SINGLE STRAND, PO 4, RIBOSE SUGAR. q BASE PAIRS (N) ü T=THYMINE ü A=ADENINE ü C= CYTOSINE ü G=GUANINE q BASE PAIRS (N) ü U = URACIL ü A=ADENINE ü C=CYTOSINE ü G=GUANINE 2

URACIL (U) base with a single-ring structure phosphate group sugar (ribose) 3

POINTS ABOUT TRANSCRIPTION ü NEED RNA POLYMERASE ü CODES FOR 20 AMINO ACIDS ü CODON: SERIES OF TRIPLET BASE PAIRS. ü 64 CODONS, 60 FOR AA, OTHERS FOR STARTS/STOPS. ü INTRONS=NON-CODING ü EXONS= CODING FOR RNA 4

PROTEIN TRANSCRIPTION ü NUCLEUS ü RNA POLYMERASE CODES TO DNA ü DNA TRANSCRIBES TO m-RNA ü INTRONS SNIPPED OUT ü EXONS KEPT IN CODE 5

exon unit of transcription in a DNA strand intron exon 3’ 5’ transcription into pre-m. RNA poly-A tail cap 5’ 3’ (snipped out) 5’ 3’ mature m. RNA transcript 6

sugar-phosphate backbone of one strand of nucleotides in a DNA double helix sugar-phosphate backbone of the other strand of nucleotides part of the sequence of base pairs in DNA transcribed DNA winds up again DNA to be transcribed unwinds Newly forming RNA transcript The DNA template at the assembly site growing RNA transcript 5’ 3’ 5’ direction of transcription 3’ 5’ 3’ RNA polymerase 7

PROTEIN TRANSLATION ü m-RNA GOES THRU RIBOSOME. ü RIBOSOME IS r. RNA, CODE THREADS THRU RIBOSOME. ü AREA OF RIBOSOME BOUND TO t. RNA ü 20 TYPES OF AA ü ANTICODON ON ONE END OF t-RNA. ü AA ON OTHER END OF t-RNA ü AA ATTACH TO EACH OTHER IN PEPTIDE BOND ü FORM PROTEINS 8

Binding site for m. RNA P (first binding site for t. RNA) A (second binding site for t. RNA) 9

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TRANSCRIPTION Pre m. RNA Transcript Processing Unwinding of gene regions of a DNA molecule m. RNA r. RNA t. RNA protein subunits Mature m. RNA transcripts TRANSLATION Synthesis of a polypetide chain at binding sites for m. RNA and t. RNA on the surface of an intact ribosome ribosomal subunits mature t. RNA Convergence of RNAs Cytoplasmic pools of amino acids, t. RNAs, and ribosomal subunits FINAL PROTEIN Destined for use in cell or for transport 11

VALINE PROLINE THREONINE LEUCINE HISTIDINE GLUTAMATE VALINE PROLINE THREONINE VALINE LEUCINE HISTIDINE GLUTAMATE 12

m. RNA transcribed from the DNA PART OF PARENTAL DNA TEMPLATE ARGININE GLYCINE TYROSINE TRYPTOPHAN ASPARAGINE ARGININE GLYCINE LEUCINE GLUTAMATE resulting amino acid sequence altered message in m. RNA A BASE INSERTION (RED) IN DNA the altered amino acid sequence 13

Overview: the roles of transcription and translation in the flow of genetic information 14

The triplet code 15

TRANSCRIPTION AND TRANSLATION ü C DNA. ü m-RNA. ü t-RNA. ü AMINO ACID ATC-GCG-TAT UAG-CGC-AUA AUC-GCG-UAU ISO-ALA-TYR ü PEPTIDE BONDS/POLYPEPTIDES/PROTEINS 16

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Translation Nuclear membrane DNA Transcription Eukaryotic Cell Pre-m. RNA Processing m. RNA Ribosome Translation Protein 18

Translation ü Synthesis of proteins in the cytoplasm ü Involves the following: 1. m. RNA (codons) 2. t. RNA (anticodons) 3. r. RNA 4. ribosomes 5. amino acids 19

Types of RNA ü Three types of RNA: RNA A. messenger RNA (m. RNA) B. transfer RNA (t. RNA) C. ribosome RNA (r. RNA) ü Remember: all produced in the nucleus! 20

A. Messenger RNA (m. RNA) ü Carries the information for a specific protein ü Made up of 500 to 1000 nucleotides long. ü Made up of codons (sequence of three bases: AUG - methionine). ü Each codon, codon is specific for an amino acid 21

A. Messenger RNA (m. RNA) start codon m. RNA A U G G G C U C C A U C G G C A U A A codon 1 protein methionine codon 2 codon 3 glycine serine codon 4 isoleucine codon 5 codon 6 glycine alanine codon 7 stop codon Primary structure of a protein aa 1 aa 2 aa 3 peptide bonds aa 4 aa 5 aa 6 22

B. Transfer RNA (t. RNA) ü Made up of 75 to 80 nucleotides long. ü Picks up the appropriate amino acid floating in the cytoplasm (amino acid activating enzyme) enzyme ü Transports amino acids to the m. RNA ü Have anticodons that are complementary to m. RNA codons ü Recognizes the appropriate codons on the m. RNA and bonds to them with H-bonds. 23

anticodon in m. RNA anticodon t. RNA MOLECULE amino acid attachment site OH amino acid attachment site 24

The structure of transfer RNA (t. RNA) 25

B. Transfer RNA (t. RNA) amino acid attachment site methionine U A amino acid C anticodon 26

C. Ribosomal RNA (r. RNA) ü Made up of r. RNA is 100 to 3000 nucleotides long. ü Important structural component of a ribosome. ü Associates with proteins to form ribosomes. 27

Ribosomes ü Large and small subunits. ü Composed of r. RNA (40%) and proteins (60%). ü Both units come together and help bind the m. RNA and t. RNA. ü Two sites for t. RNA a. P site (first and last t. RNA will attach) attach b. A site 28

Ribosomes Origin Cytosol (eukaryotic ribosome) Chloroplasts (prokaryotic ribosome) Complete Ribosomal ribosome subunit 80 S 40 S 60 S r. RNA components 18 S 5 S 5. 8 S 25 S Proteins 70 S 30 S 50 S 16 S 4. 5 S 23 S C. 24 C. 35 30 S 50 S 18 S 5 S 26 S C. 33 C. 35 Mitochondrion 78 S (prokaryotic ribosome) C. 30 C. 50 29

Ribosomes Large subunit P Site A Site m. RNA A U G Small subunit C U A C U U C G 30

Translation ü Three parts: 1. initiation: initiation start codon (AUG) 2. elongation: elongation 3. termination: termination stop codon (UAG) ü Let’s make a PROTEIN!!!! 31

Translation Large subunit P Site A Site m. RNA A U G Small subunit C U A C U U C G 32

Translation • Initiation The inactive 40 S and 60 S subunits will bind to each other with high affinity to form inactive complex unless kept apart This is achieved by e. IF 3, which bind to the 40 S subunit m. RNA forms an initiation complex with a ribosome A number of initiation factors participate in the process. 33

Translation ü Cap sequence present at the 5’ end of the m. RNA is recognized by e. IF 4 ü Subsequently e. IF 3 is bound and cause the binding of small 40 S subunit in the complexes ü The 18 S RNA present in the 40 S subunit is involved in binding the cap sequence ü e. IF 2 binds GTP and initiation t. RNA, which recognize the start codon AUG ü This complex is also bound to 40 S subunit 34

Translation ü Driven by hydrolysis of ATP, 40 S complex migrate down stream until it finds AUG start codon ü The large 60 S subunit is then bound to the 40 S subunit ü It is accompanied by the dissociation of several initiation factor and GDP ü The formation of the initiation complex is now completed ü Ribosome complex is able to translate 35

Translation ü Extrachromosomal m. RNAs have no cap site ü Plastid m. RNA has a special ribosome binding site for the initial binding to the small subunit of the ribosome (shine-Dalgarno sequence) ü This sequence is also found in bacterial m. RNA, but it is not known in the mitochondria ü In the prokaryotic, the initiation t. RNA is loaded with Nformylmethionine ü After peptide formation, the formyl residue is cleaved from the methionine 36

Initiation aa 1 1 -t. RNA anticodon hydrogen bonds U A C A U G codon aa 2 2 -t. RNA G A U C U A C U U C G A m. RNA 37

Translation • Elongation A ribosome contains two sites where the t. RNAs can bind to the m. RNA. P (peptidyl) site allows the binding of the initiation t. RNA to the AUG start codon. The A (aminoacyl) site covers the second codon of the gene and the first is unoccupied On the other side of the P site is the exit (E) site where empty t. RNA is released 38

Translation • Elongation The elongation begins after the corresponding aminoacyl-t. RNA occupies the A site by forming base pairs with the second codon Two elongation factors (e. EF) play an important role e. EF 1 binds GTP and guides the corresponding aminoacyl-t. RNA to the A site, during which GTP is hydrolized to GDP and P. The cleavage of the energy-rich anhydride bond in GTP enables the aminoacyl-t. RNA to bind to codon at the A site 39

Translation • Elongation Afterwards the GDP still bound to e. EF 1 , is exchange for GTP as mediated by the e. EF 1 The e. EF 1 -GTP is now ready for the next cycle Subsequently a peptide linkage is form between the carboxyl group of methionine and the amino group of amino acid of the t. RNA bound to A site Peptidyl transferase catalyzing the reaction. It facilitates the N-nucleophilic attack on the carboxyl group, whereby the peptide bond is formed with the released of water 40

Translation • Elongation Accompanied by the hydrolysis of one molecule GTP to form GDP and P, the e. EF 2 facilitates the translocation of the ribosome along the m. RNA to three bases downstream Free t. RNA arrives at site E is released, and t. RNA loaded with the peptide now occupies the P Site The third aminoacyl-t. RNA binds to the vacant A site and a further elongation cycle can begin 41

Elongation peptide bond aa 3 aa 1 aa 2 3 -t. RNA 1 -t. RNA anticodon hydrogen bonds U A C A U G codon 2 -t. RNA G A U C U A C U U C G A m. RNA 42

aa 1 peptide bond aa 3 aa 2 1 -t. RNA 3 -t. RNA U A C (leaves) 2 -t. RNA A U G G A A G A U C U A C U U C G A m. RNA Ribosomes move over one codon 43

aa 1 peptide bonds aa 2 aa 4 aa 3 4 -t. RNA 2 -t. RNA A U G 3 -t. RNA G C U G A A C U U C G A A C U m. RNA 44

aa 1 peptide bonds aa 4 aa 2 aa 3 2 -t. RNA 4 -t. RNA G A U (leaves) 3 -t. RNA A U G G C U G A A C U U C G A A C U m. RNA Ribosomes move over one codon 45

aa 1 peptide bonds aa 5 aa 2 aa 3 aa 4 5 -t. RNA U G A 3 -t. RNA 4 -t. RNA G A A G C U A C U U C G A A C U m. RNA 46

peptide bonds aa 1 aa 5 aa 2 aa 3 aa 4 5 -t. RNA U G A 3 -t. RNA G A A 4 -t. RNA G C U A C U U C G A A C U m. RNA Ribosomes move over one codon 47

aa 4 aa 5 Termination aa 199 aa 3 primary structure aa 2 of a protein aa 200 aa 1 200 -t. RNA A C U terminator or stop codon C A U G U U U A G m. RNA 48

Translation • Release ü When A site finally binds to a stop codon (UGA, UAG, UAA) ü Stop codons bind e. RF accompanied by hydrolysis GTP to form GDP and P ü Binding of e. RF to the stop codon alters the specificity the peptidyl transferase ü Water instead amino acid is now the acceptor for the peptide chain ü Protein released from the t. RNA 49

Translation • The difference • Eukaryotic and prokaryotic translation can react differently to certain antibiotics Puromycin an analog t. RNA and a general inhibitor of protein synthesis Cycloheximide only inhibits protein synthesis by eukaryotic ribosomes Chloramphenicol, Tetracycline, Streptomycin inhibit protein synthesis by prokaryotic ribosome 50

End Product ü The end products of protein synthesis is a primary structure of a protein ü A sequence of amino acid bonded together by peptide bonds aa 2 aa 1 aa 3 aa 4 aa 5 aa 199 aa 200 51

Polyribosome • Groups of ribosomes reading same m. RNA simultaneously producing many proteins (polypeptides). incoming large subunit 1 incoming small subunit 2 3 4 polypeptide 5 6 7 m. RNA 52

TYPES OF PROTEINS ü ENZYMES/HELICASE ü CARRIER/HEMOGLOBIN ü IMMUNOGLOBULIN/ANTIBODIES ü HORMONES/STEROIDS ü STRUCTURAL/MUSCLE ü IONIC/K+, Na+ ü all regulate things put together ”critter” 53

Protein Sorting ü Vast majority of protein within the cell are synthesized within the cytoplasm, but the final sub-cellular location can be in one of a whole array of membrane-bound compartment ü Protein is subjected to be sorted for special targeted organelles 54

Protein Sorting ü Vast majority of protein within the cell are synthesized within the cytoplasm, but the final sub-cellular location can be in one of a whole array of membrane-bound compartment ü Protein is subjected to be sorted for special targeted organelles: Plastids Mitochondria Peroxisomes Vacuoles 55

Mitochondria ü More than 95% of mitochondrial proteins in plant are encoded in the nucleus and translated in the cytosol ü Proteins are generally equipped with targeting signals ( a signal sequence of 12 -70 amino acids at the amino terminal) ü Protein import occurs at translocation site ü In most cases, protein destined for the mitochondrial inner membrane after transport through outer membrane are guided directly to the location by internal targeting sequence ü Protein destined for the inner mitochondrial membrane contain prosequence that guides first into the mitochondrial matrix. After removal of the pro-sequence by processing peptidase, the proteins are directed by second targeting signal sequence into the inner membrane 56

Plastids ü ATP is consumed for the phosphorilation of a protein, probably the receptor OEP 86 ü The protein transport is regulated by the binding of the GTP to OEP 86 and OEP 34 ü After the protein is delivered, the pre-sequence is removed by a processing peptidase ü The protein destined to thylakoid membrane are first delivered into stroma and then directed by internal targeting signal into thylakoid membrane 57

Peroxisomes ü Small membrane-bound cytoplasmic organelle containing oxidizing enzymes ü They can be found in leaf cells where they contain some of the enzymes of glycolytic pathway ü All protein have to be delivered from the cytosol ü The transport is accompanied by ATP hydrolysis ü Targeting sequence SKL (serine-lysine-leucine) has been observed in C terminus, but this sequence is not removed after uptake 58

Vacuole ü Proteins are transferred during their synthesis to the lumen of ER ü This is aided by a signal sequence at the terminus of the synthesized protein, which binds with a signal recognition particle to a pore protein present in the ER membrane and thus directs the protein to the ER lumen ü In such cases, ribosome is attached to the ER membrane during protein synthesis and the synthesized protein appears immediately in the ER lumen. It is called co-translational protein transport ü This protein is then transferred from the ER by vesicles transfer across the golgi apparatus to the vacuole or are exported by secretory vesicles from the cell 59

Coupled transcription and translation in bacteria 60

original base triplet in a DNA strand a base substitution within the triplet (red) As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it: POSSIBLE OUTCOMES: OR One DNA molecule carries the original, unmutated sequence VALINE PROLINE The other DNA molecule carries a gene mutation THREONINE VALINE LEUCINE HISTIDINE 61 GLUTAMATE

A summary of transcription and translation in a eukaryotic cell 62