Lecture 3: Transcription (Cont), Genetic Code and Translation Learning Objectives

Define a codon and the genetic code

• Codon: A specific sequence of three consecutive nucleotides in mRNA that specifies a single amino acid or signals the end of translation during protein synthesis

• Genetic code: The sequence of nucleotides, coded in triplets (codons) along the mRNA that determines the sequence of amino acids in proteins

List the key characteristics of the genetic code and illustrate each with an example

• The triplet code is colinear and nonoverlapping.

1. Ex: In 5’-AUG-GUC-UCA-3’ bases 1-3 (AUG) code for Methionine and bases 4-6 (GUC) code for Valine. Base 3 (G0) is never re-used to to form an overlapping codon like UGG

• The triplet code is redundant (aka degenerate) meaning multiple codons can code for the same amino acid

1. Ex: Leucin is coded by six different codons (UUA, UUG, CUU, CUC, CUA, and CUG)

• The triplet code contains one codon to initiate translation and three codons to stop translation

1. The codon AUG (Methionine) is the start codon for initiating translation and codons UUA, UAG, and UGA are the three stop codons to terminate transcription

• The triple code is unambiguous meaning that each specific codon codes for one, and only one, amino acid or signal

1. Ex: The codon CAG only codes for the amino acid Glutamine and no other amino acid

• The triplet code does not contain breaks between codons

1. Ex: In the sequence 5’-AUG-CCC-GGG-3', the ribosome reads AUG, immediately shifts to CCC, and directly transitions to GGG. If a single base is deleted, the reading frame shifts completely (frameshift mutation) rather than skipping the gap

• The triplet code is (almost) universal

1. Ex: Human mRNA inserted into E. coli is read using the exact same codons to yield human protein. (Minor exceptions exist, such as human mitochondrial DNA using $\text{UGA}$ for Tryptophan instead of Stop)

Review experimental results that cracked the genetic code

• In vitro synthesis of polypeptides with synthetic mRNAs (Nirenberg and Matthaei 1961). Radio-labeled amino acids were incorporated into a polypeptide based on sequence in synthetic mRNA

• Further experiments by Khorana showed that synthesizing mRNAs with variation in sequence produced different amino acids. Still some ambiguity with this method

• Nirenberg and Leder (1965) created mRNAs with only 3 nucleotides and added these to a system with radio-labeled tRNAs. Only one radio-labeled tRNA in each version of the system. Translation extracts were filtered and trapped radioactivity indicated which amino acid was encoded by the short mRNA

Explain how different types of DNA mutations affect the genetic code and the subsequent changes that may or may not be found in the protein. Key point: codon reading frame

• Mutations in the DNA may or may not have a significant impact on the outcome of the final protein

• Substitution mutations: Replacement of one nucleotide for another. May take advantage of the wobble rule (describe how the first base of a transfer RNA (tRNA) anticodon can form flexible, non-standard base pairs with the third base of a messenger RNA (mRNA) codon during protein) and get the same amino acid. Effects may be lessened because the reading frame is still intact

• Frameshift mutations: Insertion or deletion of one or more nucleotides. The initiation site sets the reading frame, and codons must be read as triplets

List and describe the required components of protein synthesis (aka translation)

• Translation (protein synthesis) is the process that uses the sequence of nucleotides in an mRNA to direct assembly of the sequence of amino acids in the polypeptide

• Translation has 3 required components:

1. Fully processed mRNA

2. Ribosomes

3. Transfer RNA (tRNA)

• mRNA:

1. Template to make the protein product

2. A copy of the DNA gene sequence

• Two untranslated regions (UTRs) are present in final message (eukaryotes only):

1. 5’ UTR

2. 3’ UTR

• Ribosomes:

1. Complexes of rRNA and protein with enzymatic activity

2. Functioning ribosome has a large and small subunit. Small subunit binds mRNA. Large subunit binds tRNAs

3. Catalyzes the peptide bond between amino acids

• When assembled, ribosomes have three sites:

1. Aminoacyl (A) site-binds incoming tRNAs

2. Peptidyl (P) site-peptide bond formation

3. Exit (E) site-tRNA molecules leave the ribosome

• Transfer RNA (tRNA):

1. A short, non-coding RNA molecule

2. Forms a structure called a cloverleaf

3. tRNAs are the molecules that “translate” between mRNA and proteins

4. Contain the anticodon and a binding site for each amino acid

5. tRNA molecules must be “charged” for translation

6. The enzymes that pair tRNA with amino acids are aminoacyl tRNA synthetases

7. Must be specific for the amino acid and anticodon of the tRNA

8. Pairing corresponding amino acids and tRNA molecules is key for the accuracy of translation

9. The interaction that determines the incorporation of the amino acid into the polypeptide is between the codon and anticodon

List and describe the major stages of protein synthesis: initiation, elongation, termination

• Initiation:

1. Initiation requires: mRNA, Charged tRNA with Met, Small ribosomal subunit, Initiation factor proteins

2. To properly align the ribosome, the 5’ cap and 3’tail are used along with initiation factors. The consensus Kozak sequence contains the start codon

3. Pre-initiation complex scans mRNA seeking first AUG codon within Kozak sequence

4. AUG codon recognition causes dissociation of eIF factors and recruits large ribosomal subunit

5. Reading frame for translation is now set

6. Binding of the 3’ polyA tail circularizes the transcript. Protects mRNA from degradation

• Elongation:

1. Initiator tRNA binds to P site, subsequent tRNAs are bound to A site

2. Formation of peptide bonds via peptidyl transferase

3. “Empty” tRNAs leave the ribosome via the E site

4. Direction of peptide synthesis is N terminus to C terminus

5. New tRNAs are brought to A site with elongation factor eEF-1A. eEF-1A carries GTP and serves as a “clock” for elongation

6. Elongation factors that travel with charged tRNAs also have GTP

7. The hydrolysis of GTP to GDP with these factors serves as a check on correct codon-anticodon pairing. If pairing is correct, GTP is hydrolyzed relatively quickly. If pairing is incorrect, GTP hydrolysis is recognized as “too slow”, and incorrect tRNA is removed

• Terminaton:

1. Requires stop codons and release factors

2. Only release factors recognize the stop codons. When these enter the A site, they hydrolyze the polypeptide from the tRNA in the P site. Eukaryotes have only one release factor

3. When translation is complete, all components dissociate

Describe the key regulatory steps of each stage of translation