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Biol115 The Thread of Life Lecture 7 Lost in translation: mRNA translation & protein synthesis “If the dream is a translation of waking life, waking life is also a translation of the dream.” ~ Rene Magritte Principles of Biology • Chapter ‘Gene Expression’ • Chapter ‘Translation’ Biol115_2014_Lecture 7 2 Objectives • Describe the molecular structures involved in translation. • Explain the process of translation in detail. • Explain how newly synthesised proteins are delivered to different destinations (membranes and extracellular space). • Key terms: anticodon, elongation factor, initiation factor, polyribosome, release factor, ribosome, signal peptide, signalrecognition particle (SRP), transfer RNA (tRNA) Biol115_2014_Lecture 7 3 Basic principles of translation • Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA • Ribosomes are the sites of translation • In a eukaryotic cell, the nuclear envelope separates transcription from translation Biol115_2014_Lecture 7 4 Translation Conversion from nucleotides, the “language” of mRNA, to amino acids, the “language” of proteins. Biol115_2014_Lecture 7 5 Codons: triplets of bases • There are 20 amino acids, but there are only four nucleotide bases in DNA • The flow of information from gene to protein is based on a triplet code: a series of non-overlapping, three-nucleotide words (codons) • Therefore, 43 (= 64) codons to specify 64 amino acids. Biol115_2014_Lecture 7 6 Cracking the code • All 64 codons were deciphered by the mid-1960s • Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation • One codon, AUG, is the universal “start” codon. • The genetic code is redundant but not ambiguous; no codon specifies more than one amino acid The amino acids specified by each mRNA codon. Multiple codons can code for the same amino acid. The codons are written 5' to 3', as they appear in the mRNA. AUG is an initiation codon; UAA, UAG, and UGA are termination (stop) codons. Biol115_2014_Lecture 7 7 Evolution of the genetic code • The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals • Genes can be transcribed and translated after being transplanted from one species to another Biol115_2014_Lecture 7 8 Molecular components of translation • mRNA: A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) • tRNA: Molecules of tRNA are not identical: • Each carries a specific amino acid on one end • Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA • Ribosomes and ribosomal RNAs Biol115_2014_Lecture 7 9 The structure and function of transfer RNA A C C • A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long • Contains an amino acid attachment site and an anticodon that recognizes the complementary codon on the mRNA. Biol115_2014_Lecture 7 10 Transfer RNA (tRNA) Adapter molecules that recognize codons in the mRNA and link them to specific amino acids. Biol115_2014_Lecture 7 11 Ribosomes • Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis • The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA) Biol115_2014_Lecture 7 12 The amazing complexity of ribosomes http://bass.bio.uci.edu/~hudel/bs9 9a/lecture22/ribosome70s_a.gif http://bass.bio.uci.edu/~hudel/bs99a/lecture22/exit_tunnel.jpg Biol115_2014_Lecture 7 13 Ribosomes • A ribosome has three binding sites for tRNA: • The P site holds the tRNA that carries the growing polypeptide chain • The A site holds the tRNA that carries the next amino acid to be added to the chain • The E site is the exit site, where discharged tRNAs leave the ribosome tRNAs move through these sites in this order. Biol115_2014_Lecture 7 14 Building a polypeptide • The three stages of translation: • Initiation • Elongation • Termination • All three stages require protein “factors” that aid in the translation process Biol115_2014_Lecture 7 15 A gene is expressed through the processes of transcription and translation. During transcription, the enzyme RNA polymerase (green) uses DNA as a template to produce a pre-mRNA transcript (pink). The pre-mRNA is processed to form a mature mRNA molecule that can be translated to build the protein molecule (polypeptide) encoded by the original gene. Biol115_2014_Lecture 7 16 A DNA transcription unit A DNA transcription unit is composed, from its 3' to 5' end, of an RNA-coding region (pink rectangle) flanked by a promoter region (green rectangle) and a terminator region (black rectangle). Regions to the left, or moving towards the 3' end, of the transcription start site are considered "upstream" regions to the right, or moving towards the 5' end, of the transcription start site are considered "downstream." Biol115_2014_Lecture 7 17 Translation Begins After the Assembly of a Complex Structure When translation begins, the small subunit of the ribosome and an initiator tRNA molecule assemble on the mRNA transcript. The small subunit of the ribosome has three binding sites: an amino acid site (A), a polypeptide site (P), and an exit site (E). The initiator tRNA molecule carrying the amino acid methionine binds to the AUG start codon of the mRNA transcript at the ribosome’s P site where it will become the first amino acid incorporated into the growing polypeptide chain. Here, the initiator tRNA molecule is shown binding after the small ribosomal subunit has assembled on the mRNA; the order in which this occurs is unique to prokaryotic cells. In eukaryotes, the free initiator tRNA first binds the small ribosomal subunit to form a complex. The complex then binds the mRNA transcript, so that the tRNA and the small ribosomal subunit bind the mRNA simultaneously. Biol115_2014_Lecture 7 18 Ribosome association and initiation of translation • The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits • First, a small ribosomal subunit binds with mRNA and a special initiator tRNA • Then the small subunit moves along the mRNA until it reaches the start codon (AUG) • Proteins called initiation factors bring in the large subunit that completes the translation initiation complex Biol115_2014_Lecture 7 19 Insert Fig. 17-17 P342 Biol115_2014_Lecture 7 20 Elongation of the polypeptide chain • During the elongation stage, amino acids are added one by one to the preceding amino acid • Each addition involves proteins called elongation factors and occurs in three steps: codon recognition, peptide bond formation, and translocation Biol115_2014_Lecture 7 21 Insert Fig. 17-18 P343 Biol115_2014_Lecture 7 22 Termination of translation • Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome • The A site accepts a protein called a release factor • The release factor causes the addition of a water molecule instead of an amino acid • This reaction releases the polypeptide, and the translation assembly then comes apart Biol115_2014_Lecture 7 23 Insert Fig. 17-19 P344 Occurs when a stop codon is encountered. Release factors bind to the stop codon and break the bond between the tRNA and the polypeptide. Biol115_2014_Lecture 7 24 Polyribosomes • A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome) • Polyribosomes enable a cell to make many copies of a polypeptide very quickly Biol115_2014_Lecture 7 25 Targeting polypeptides to specific locations • Two populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER) • Free ribosomes mostly synthesise proteins that function in the cytosol • Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell • Ribosomes are identical and can switch from free to bound Biol115_2014_Lecture 7 26 Targeting polypeptides to specific locations • Polypeptide synthesis always begins in the cytosol • Synthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER • Polypeptides destined for the ER or for secretion are marked by a signal peptide • A signal-recognition particle (SRP) binds to the signal peptide • The SRP brings the signal peptide and its ribosome to the ER Biol115_2014_Lecture 7 27 Insert Fig. 17-21 P345 Biol115_2014_Lecture 7 28 You should now be able to: 1. Compare transcription and translation in bacteria and eukaryotes. 2. Explain what it means to say that the genetic code is redundant and unambiguous. 3. Include the following terms in a description of translation: tRNA, wobble, ribosomes, initiation, elongation, and termination. Biol115_2014_Lecture 7 29