Protein Synthesis

Central Dogma

Transcription - mRNA Genetic information is first transcribed into an RNA molecule. This intermediary RNA molecule is called messenger RNA (mRNA) DNA serves as the template for the synthesis of RNA - much as it does for its own replication. 7/7/20163

Transcription Overview 7/7/20164

Transcription - promoter site RNA Polymerase will bind to the promoter site on the DNA The promoter site includes the initiation site plus other important sites ie TATA box RNA Polymerase is just one component of a transcription “machine” consisting of around 100 different components. 7/7/20165

Transcription - production of mRNA strand Transcription complex untwists one turn of the DNA double helix at a time It proceeds down one strand moving in the 3' - 5' direction As it does so, each ribonucleotide is inserted into the growing RNA strand following the rules of base pairing. For each C a G and for each G a C, for each T, an A. 7/7/20166

Transcription - Uracil An “A” on the DNA causes the insertion of the pyrimidine uracil (U) RNA polymerase links together As the RNA nucleotides RNA strand forms at 60 nucleotides per second, 5 million every 24 hours 7/7/20167

Transcription - continued Hydrogen bonds reform between separated DNA strand as RNA strand peels away. A single gene can be transcribed simultaneously by several molecules of RNA polymerase, following each other like trucks in a convoy 7/7/20168

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Transcription - termination Transcription stops after the RNA polymerase has moved the length of the gene and come into contact with the terminator sequence. 7/7/201610

Termination The most common termination sequence in Eukaryotes is AATAAA At this sequence the RNA polymerase releases the mRNA and detaches from the gene. 7/7/201611

Which DNA Strand is transcribed? A promoter signals only one strand to be transcribed However, both strands of the DNA serve as templates for different loci Some genes are coded for by one strand and some by the other. The entire stretch of DNA that is transcribed into a single RNA molecule is called a Transcription Unit 7/7/201612

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Eukaryotic and Prokaryotic Transcription Units In Eukaryotes a transcription Unit represents a single gene In Prokaryotes a Transcription unit may include a few genes -see operon theory later Bacteria have a single type of RNA polymerase Eukaryotes have three types of RNA polymerase enzyme 7/7/201614

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tRNA - shuttle molecule tRNA is transcribed from the DNA like other types of RNA It travels to the cytoplasm, each tRNA can be used repeatedly Some of RNA sequence is common to all RNA some unique About 20 types 7/7/201616

tRNA Consists of a single RNA strand about 80 nucleotides long Strand folds back to form a complex 3D structure - see diagram One important region is the anticodon - the specialized base triplet that binds to the mRNA codon The 3’ end is the amino acid attachment site – always CCA 7/7/201617

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Attachment of Amino Acids to tRNA Attachment of the Amino Acid to tRNA is brought about by aminoacyl tRNA synthetase enzyme and requires energy There are at least 20 specific synthetase enzymes - one for each type of tRNA 7/7/201619

Overview of Translation 7/7/201620

Translation Transfer of information from one language (nucleotides) to another (amino acids) Three stages – Initiation – Elongation – Termination 7/7/201621

Initiation Begins when smaller ribosomal subunit and a special initiator tRNA molecule attaches to a strand of mRNA The mRNA bonds to the small ribosomal subunit in the presence of initiation factors. The start codon (AUG) of mRNA binds to the anticodon of the initiator tRNA (f met) at the same time. This is known as the initiation complex 7/7/201622

7/7/ Ribosome structure A P P P P P P P P P-site peptidyl tRNA site A-site aminoacyl tRNA site mRNA5’5’ Small subunit Large subunit Ribosome with bound tRNAs and mRNA

7/7/ Large ribosomal subunit Protein (purple) lies on the surface 23S RNA (orange and white) makes up the core of the subunit

7/7/ mRNA5’ cap 40S subunit M AUG Initiator tRNA bound to the small ribosomal subunit Initiation – step 1 Initiation – step 1 : mRNA binding The small subunit finds the 5’ cap and scans down the mRNA to the first AUG codon

Initiation - step 2 Large ribosomal subunit binds to the small one to form a functional ribosome. The initiator tRNA becomes locked into the P site on the ribosome 7/7/201626

7/7/ mRNA5’5’ 40S subunit M AUG the initiation codon is recognized the large ribosomal subunit binds 60S subunit

7/7/ mRNA5’5’ M AUG aminoacyl tRNA binds the A-site first peptide bond is formed initiation is complete GCC A mRNA5’5’ M AUGGCC A

Elongation - step 1 Second codon of the mRNA is positioned opposite the A (aminoacyl) site of the large subunit Hydrogen bonds are formed between the anticodon of the tRNA entering the A site and the second codon of the mRNA Several proteins called elongation factors are involved in the process of elongation 7/7/201629

Elongation - Step 2 In the second step, peptidyl transferase catalyzes the formation of a peptide bond between the polypeptide in the P site and the new amino acid in the A site. 7/7/201630

Elongation - step 3 Translocation The polypeptide then separates from the tRNA to which it was bound and is transferred to the amino acid carried by the tRNA in the A site. The tRNA occupying the P site separates from the ribosome and the tRNA in the A site is translocated to the P site. 7/7/201631

Translocation - continued The codon and anticodon remain bonded allowing the mRNA and the tRNA to move as a unit. This movement brings the next codon to be translated into the A site. Each step in the process requires energy. 7/7/201632

Elongation Cycle The mRNA is moved through the ribosome only in the 5' to 3'direction. The elongation cycle is repeated until a termination codon reaches the A site. 7/7/201633

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Termination A termination codon is a signal to stop translation. When these codons reach the A site of the ribosome, a protein called release factor binds to the codon. Examples are UAA, UAG, UGA The release factor causes peptidyl transferase to add a water molecule to the polypeptide chain instead of an amino acid. 7/7/201636

Termination - continued This reaction frees the completed polypeptide from the tRNA in the P site. The ribosome then separates back into a small subunit and a large subunit. 7/7/201637

7/7/ P UCAGCA GGG UAG P P P P Termination when translation reaches the stop codon, a release factor (RF) binds within the A-site, recognizing the stop codon release factor catalyzes the hydrolysis of the completed polypeptide from the peptidyl tRNA, and the entire complex dissociates RF P UCAGCA GGG UAG P P P P P P P

7/7/ The Triplet Code

The Codon Reading Frame 7/7/201640