Chapter 14 translation

genetics

Polypeptide

Long chain of amino acids linked by peptide bonds

Peptide bonds

Covalent bond that forms been the carboxyl end of an amino acid and the amino acid of the next bond

Calmodulin

• Present in all eukaryotes

• A small protein that is a single polypeptide chain with an overall tertiary structure

• Has alpha helices (secondary structure)

Hemoglobin

• The protein inside red blood cells that carries oxygen in the bloodstream

• Has 4 polypeptides (2 a-globin’s and b-globin’s) making it a quaternary structure

Structural organization of Hemoglobin 

1. Primary structure 

• Polypeptide: a sequence of amino acids

2. Secondary structure

• Alpha helix: Partial folding

3. Tertiary structure

• Globin: Complete folded polypeptide 

4. Quaternary structure 

• Hemoglobin: 4 globin’s 

Codon

A sequence of 3 nucleotides of DNA that code for an amino acid

• mRNA codons-  5’ to 3’ direction  

Starting codon

AUG (methionine)

Ending codon

UAA, UAG, UGA (these DON’T code for an amino acid)

Reading frame

A linear sequence of codons in a nucleic acid defined by a start codon and a stop codon

Characteristics of the genetic code 

1. Unambiguous 

• Each of the 61 triplets code for only one of the 20 amino acids

2. Degenerate

• Most amino acids are encoded by more than one codon

3. Universal

• MOST living organisms use the same code but there are exceptions

4. Commaless

• No breaks between the codons in a reading frame (read continuously as groups of 3)

5. Non- overlapping

• Triplets in a reading frame are in a tandem sequence and don’t overlap 

Central Dogma (The flow of genetic code)

DNA → RNA → Protein

• DNA is the long-term storage of genetic information.

• RNA is the temporary copy used to build proteins.

• Proteins do the work in the cell and determine traits.

The flow of genetic code (Central dogma)

1. DNA (Template/ antisense strand)

• The 3’ to 5’ DNA template strand is used to make mRNA

2. Transcription

• RNA polymerase reads the DNA template strand and builds mRNA in the 5’ to 3’ direction

3. Translation

• Ribosomes read the mRNA codons (in groups of three) and converts them into a chain of amino acids 

4. Protein (polypeptide)

• Amino acids link to form a protein which preforms functions in the cell

Spontaneous mutations

DNA inside the cell can be exposed to radiations and molecules that react with it and cause chemical alterations (some of which can be permanent) 

Substitution mutations 

A base replaced by a different base in DNA causes a permanent single codon change.

EX. 

• Silent: The resulting new codon codes for the same amino acid as the original one

• Missense: The new codon codes for a different amino acid 

• Nonsense: the new codon is a stop codon, resulting in a shorter polypeptide 

Note:

• Mutations can be cause by errors in DNA replication and can be copied into mRNA’s

Insertion of a single base results in frameshift mutation 

• Insertions are due to errors in DNA replication 

Frameshift mutation occurs when one base (one letter in a code) of a codon is inserted or deleted 

Trinucleotide repeat expansion

1. Starts with 8 CAG gene

2. Two strands separate

3. Then replicate

4. Hairpin forms on newly synthesized gene

5. causes part of the template strand to be replicated twice and increases the number of repeats on the newly synthesized strand

6. two strands of the DNA molecule separate

7. the strand with extra CAG serves as a template for replication

8. resulting DNA molecules have 5 extra CAG strand repeats 

Huntington’s disease Trinucleotide repeat expansion

A neurodegenerative disorder (autosomal dominant) caused by abnormal repeats of CAG gene (codes for glutamine in mRNA in the Huntington gene) resulting in polyglutamine Huntington gene

Basic requirements of translation

• mRNA 

• charges transfer RNA’s (tRNA)

• ribosome

• no primers

• other: initiation factors, elongation factors, energy sources 

Transfer RNA’s

An amino acid is attached to the 3’ end of the tRNA. The Anti-codon loop base pairs with a codon in the mRNA 

Unusual bases found in tRNA’s

• Post transcriptional modified of bases in tRNA’s result in unusual bases

Inosine: a modified adenine 

Inosine 

a post transcriptionally modified adenine that can occur at the wobble site of tRNA’s 

The wobble hypothesis 

The interaction between the 3rd position of the codon in the mRNA and the 1st position of the anticodon in the tRNA 

• Some tRNA bases can pair with multiple mRNA bases at the wobble position, allowing translation to occur without the cell needing to synthesize all 61 tRNA’s

Basically explains how one tRNA can read multiple codons as it doesn’t need a perfect base pairing 

why one tRNA can read multiple codons for the same amino acid?

1. Pairing at the third codon position is relaxed
   The first two bases pair normally, but the third can “wobble.”

2. G in the anticodon can pair with C or U
   

• That’s why one tRNA can read multiple codons for the same amino acid.

Charging of tRNA’s (attaching an amino acid to it’s tRNA)

The carboxyl group of the amino acid is covalently attached to the 3’ end of a tRNA

• Charging is catalyzed by 20 different aminoacyl tRNA synthases

when the correct amino acid is added

1. the enzyme recognizes the amino acid by it’s R group

2. the enzyme recognizes the tRNA by it’s shape and base sequence

3. Energy is spent during this process in order to create a new peptide bond in the ribosome

Recognition of tRNA’s by aminoacyl tRNA syntheses

positions in…

Blue- Can’t be used to differentiate among tRNA’s (the same in all tRNA’s)

Red- Important for recognition of tRNA’s by one synthetase (unique in tRNA’s)

Yellow- Are used by more than one synthetase (shared in some tRNA’s)

Steps for charging tRNA when the correct amino acid is added

Step one: Amino acid activation 

The amino acid (AA) is transformed into aminoacyl adenylic acid (AA-AMP). This is the energy consuming step that requires ATP in order to create a new peptide bond 

Step two: Charging 

The AA-AMP loses its AMP and the carboxyl group of the amino acid attaches to the 3’ end of a tRNA

• This results in a aminoacyl tRNA (a charged tRNA)

The prokaryotic ribosome

Large particles of rRNA and proteins where translation occurs

Consists of 2 svedberg subunits:

• Small subunit: 30S

• Large subunit: 50S

• Complete ribosome = 70S

Svedberg units: The rate at which particles sediment in a centrifugal field (depends on weight, shape, and size) and can be used for measuring large molecules or large cell components such as ribosomes and organelles. 

Sites found in all ribosomes 

• Peptydyl  (P)

• Aminoacyl (A)

• Exit (E)

Steps in Prokaryotic translation initiation 

1. Where AUG is on the P site IF-3 bonds to the small subunit then mRNA bonds to the IF-3 small subunit

2. f-Met-tRNA (a complex forms with IF-2 and GTP) (found in prokaryotes) binds to AUG codon

3. The large ribosomal subunit joins the complex 

• The process requires GTP as an energy source + a series of initiation factors (IF proteins)

Elongation of the polypeptide chain

EF-Tu (an elongation factor) and GTP facilitate binding the second tRNA to the second codon at the A site

• The amino acid on the first tRNA is transferred and forms a peptide bond with the amino acid on the second tRNA (a dipeptide forms)

• The first tRNA moves to the E site 

• The mRNA is shifted to place the second codon in the P site and to bring the third codon into the A site

• The tRNA carrying the third amino acid binds the third codon on the A site

• The dipeptide on the second tRNA is transferred to form a tripeptide attached to the third tRNA

• The second tRNA moves to the E site with the help of EF-G and GTP

• The third codon moves to the P site

• translation continues... 

Initiation and elongation summary 

Simultaneous transcription and translation in prokaryotes

There is no nucleus so multiple ribosomes can translate the same mRNA at once creating a polyribosome 

Termination of translation

A STOP codon moves to the A site of the ribosome 

• RF1 (release factor 1) binds to stop codon 

• The GTP dependent RF3 releases the polypeptide chain from the last tRNA and the entire translation complex dissociates