BIOL 23373 General Genetics Fall 2024 Lecture 11 Translation I Lecture Notes PDF

BIOL 23373 – General Genetics Fall 2024 Lecture 11 Translation I Announcements Exam 1 was last Friday (Sept. 13). You will be able to view your submission and the answers after the last student completes the makeup exam (Wed. evening). We will d...

BIOL 23373 – General Genetics Fall 2024 Lecture 11 Translation I Announcements Exam 1 was last Friday (Sept. 13). You will be able to view your submission and the answers after the last student completes the makeup exam (Wed. evening). We will discuss class scores (mean, median, SD, distribution, etc.) in class on Friday, Sept. 20. Don’t forget that you get to drop your lowest exam score among the four hour exams and the final exam. If you want to meet (in person or via Zoom), email me 3 or 4 meeting day/time options. Tutoring Tutoring @ the CORD offers 1-on-1 and small group assistance in over 100 U of A courses. Students can meet with tutors in person or online by scheduling an appointment or accessing drop-in services. Learn more about Tutoring services and book an appointment at: https://success.uark.edu/academic-initia tives/tutoring.php Corresponding Readings Chapter sections: 14.1-14.4 The Flow of Genetic Information Transcription Synthesis of a single-stranded RNA molecule (transcript) from a DNA template (gene) This messenger RNA (mRNA) specifies the amino acid sequence of a polypeptide (protein) Translation Process of synthesizing a polypeptide using the mRNA template Mature Messenger RNA The sequence of the mRNA coding region dictates the amino acid sequence of the polypeptide. This is the region that is translated. The 5 untranslated region (5 UTR) and 3 UTR flank the translated region. 6 Translating Messenger RNA Boundaries of translation are defined by a start codon that corresponds to the N-terminus (amino terminus) of the protein and a stop codon that corresponds to the C-terminus (carboxyl terminus) 7 The Flow of Genetic Information Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Coding strand 5′ 3′ A C T G C C C A T G A G C G A C C C C T T C G G G C T C G G G G A A T G A A T C G T G A C G G G T A C T C G C T G G G G A A G C C C G A G C C C C T T A C T T A G C DNA 3′ 5′ Template strand Transcription 5′ 3′ mRNA A C U G C C C A U G A G C G A C C C C U U C G G G C U C G G G G A A U G A A U C G 5′ Untranslated Start Codon Stop 3′ Untranslated region codon codon region Anticodon Translation U A C U C G C U G G G G A A G C C C G A G C C C C U U tRNA Polypeptide NN3+ C-terminus N-terminus COO– (amino terminus) (carboxyl terminus) Met Ser Asp Pro Phe Gly Leu Gly Glu Peptide Amino bond acids The Genetic Code Translates Messenger RNA into a Polypeptide The term “genetic code” describes the correspondence between mRNA nucleotide sequences and the amino acid sequences of the resulting polypeptides During translation, tRNAs act as adaptor molecules that interpret and act on information in mRNA tRNAs have anticodons that are complementary and antiparallel to mRNA codons 9 Transfer RNAs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ Amino acid attachment site at the 3′ single-stranded 5′ region Different tRNA molecules are encoded by different Hydrogen bonds Stem-loop structure genes G G C Anticodon tRNASer carries serine (a) Two-dimensional structure of tRNA Common features 5′ 3′ single Cloverleaf structure stranded region Acceptor stem for amino acid binding Anticodon Anticodon (b) Three-dimensional structure of tRNA Charging tRNA Molecules tRNAs used during translation are called charged tRNAs, whereas tRNAs without amino acids attached are uncharged Correct charging of each tRNA molecule is critical for the integrity of the genetic code Enzymes called tRNA synthetases catalyze the addition of the correct amino acid to tRNAs 11 tRNA Synthetases tRNA synthetase contacts several points on the tRNA in the recognition process The acceptor stem of the correct tRNA fits into the active site of the enzyme The active site contains the amino acid to be added to the tRNA; ATP provides the energy for attachment tRNA synthetase uses a proofreading system to maintain a very low error rate 12 Ribosomes Are Translation Machines Ribosomes assemble strings of amino acids called polypeptides Ribosomes are composed of multiple ribosomal RNAs (rRNAs) and proteins Ribosomes translate mRNA in the 5 – 3 direction, reading each codon and assembling the amino acids in the order specified by the codons Codon = series of 3 ribonucleotides (triplet) 13 Bacterial and Eukaryotic Ribosome Structures Ribosomes in bacteria and eukaryotes perform three tasks: 1. Bind mRNA and identify the start codon where translation begins 2. Facilitate complementary base pairing of mRNA codons and the corresponding tRNA anticodons 3. Catalyze formation of peptide bonds between amino acids on the growing polypeptide chain 14 Ribosome Composition Ribosomes are composed of two subunits, the large ribosomal subunit and the small ribosomal subunit Ribosomal subunit size is measured in Svedberg units (S), a property based on size, shape, and hydration state 15 Ribosomes of E. coli Ribosomes of E. coli are the most thoroughly studied bacterial ribosomes The small subunit is 30S and contains 21 proteins and one 16S rRNA molecule The large subunit is 50S and contains 31 proteins, a small 5S rRNA, and a large 23S rRNA The fully assembled ribosome is 70S 16 Eukaryotic Ribosomes Mammalian ribosomes are the most fully characterized eukaryotic ribosomes The small (40S) subunit contains about 35 proteins and one 18S rRNA The large subunit (60S) contains 45 – 50 proteins and three rRNA molecules: 5S, 5.8S, 28S The fully assembled ribosome is 80S 17 Important Regions of Ribosomes The aminoacyl site (A site) binds a new tRNA containing an amino acid to be added to the growing polypeptide chain 18 Important Regions of Ribosomes The peptidyl site (P site) holds the tRNA to which the polypeptide is attached 19 Important Regions of Ribosomes The exit site (E site) provides an avenue for the tRNA to exit after its amino acid has been added to the polypeptide chain 20 Important Regions of Ribosomes Ribosomes also have a polypeptide channel from which the polypeptide chain emerges 21 Translation Occurs in Three Phases Translation can be divided into three phases: Initiation Elongation Termination Phases are similar in bacteria and eukaryotes, but there are some differences 22 Translation Initiation - Similarities Initiation begins when the small ribosomal subunit binds near the 5 end of the mRNA and identifies the start codon (AUG) The initiator tRNA, carrying the first amino acid of the polypeptide, binds to the start codon The large ribosomal subunit joins the small subunit to form the intact ribosome Initiation factor proteins help control ribosome formation and binding of the initiator tRNA GTP provides the energy for initiation 23 Bacterial Translational Initiation In E. coli, six molecular components come together to initiate translation: mRNA, the small ribosomal subunit, the large ribosomal subunit, the initiator tRNA, three initiation factor proteins, and GTP 24 Bacterial Translational Initiation For most of initiation, the small (30S) subunit is affiliated with an initiation factor, IF3, which prevents the small subunit from binding the large (50S) subunit The 30S-IF3 complex binds near the 5 end of the mRNA and searches for a consensus sequence 25 Bacterial Translational Initiation The preinitiation complex forms when the 16S rRNA of the 30S ribosomal subunit pairs with the Shine-Dalgarno sequence on the mRNA 26 The Shine-Dalgarno Sequence The Shine-Dalgarno sequence is a purine-rich (AG) sequence of about six nucleotides located three to nine nucleotides upstream of the start codon A complementary pyrimidine-rich (UC) sequence is found near the 3 end of the 16S rRNA This binding positions the start codon (AUG) in what will be the P site of the fully assembled ribosome 27 The Second Step of Initiation The initiator tRNA binds to the start codon where the P site will be once the ribosome is fully assembled The amino acid on the initiator tRNA is a modified amino acid, N-formylmethionine (fMet) IF2 and GTP are bound to the tRNAfMet and IF1 joins the complex to form the 30S initiation complex 28 The Final Step of Initiation In the last stage of initiation, the 50S subunit joins the 30S subunit to form the intact ribosome The union of the two subunits is driven by hydrolysis of GTP to GDP The dissociation of IF1, IF2, and IF3 accompanies the joining of the 30S and 50S subunits to create the 70S initiation complex 29 Eukaryotic Translation Initiation Eukaryotic small (40S) ribosomal subunit forms complex with eukaryotic initiation factor (eIF) proteins eIF1A, eIF3, and a charged tRNAmet bind the small subunit to form the preinitiation complex This complex is recruited to the 5 cap of mRNA 30 Later Steps of Eukaryotic Initiation The preinitiation complex joins a group of at least four eIF4 proteins that assemble at the 5 cap Together all of these components comprise the initiation complex It then uses ATP to move the small subunit along the 5 UTR in search of the start codon 31 The Final Steps of Eukaryotic Initiation The start codon (AUG) can be located because it is embedded in a consensus sequence called the Kozak sequence: 5-ACCAUGG-3 Location of the start codon leads to recruitment of the large (60S) subunit to the complex, using energy from GTP, and the dissociation of the eIF proteins 32 X