CH 15: The Genetic Code and Translation

Describe primary, secondary, tertiary, and quaternary protein structures

1. Primary: A straight chain sequence of amino acids (polypeptide chain)

2. Secondary: Polypeptide chain folds and twists; common structures include alpha helices and beta sheets.

3. Tertiary: The overall three-dimensional shape formed by the further folding and interactions of the secondary structure.

4. Quaternary: Two or more polypeptide chains associate to assemble this structure into a larger complex.

Define codon

A sequence of three nucleotides on mRNA that specifies a particular amino acid during protein synthesis. Each corresponds to either an amino acid or a stop signal, playing a crucial role in translating genetic information into proteins.

What is meant by the “degeneracy of the genetic code”?

-Degeneracy = 1 amino acid may be specified by more than one codon

-Some amino acids are carried by more than one tRNA

-Each nucleotide can have one of four possible bases (A, G, C, or U), so there are 4³= 64 possible codons, but only 20 amino acids

****Refers to the phenomenon where multiple codons can specify the same amino acid. This redundancy helps to minimize the impact of mutations and contributes to the robustness of protein synthesis.

What is meant by wobble in the code?

-Refers to the flexibility in base pairing at the third position of a codon, allowing for non-standard pairings between the codon on mRNA and the anticodon on tRNA.

-This enables a single tRNA to recognize multiple codons that specify the same amino acid, contributing to the degeneracy of the genetic code.

***Only can wobble on the 3’ position on the codon (mRNA)

What is the start (initiation) codon?

AUG

What does the start codon encode in Prokaryotes?

AUG codes for n-formyl methionine

What does the start codon encode in Eukaryotes?

AUG codes for methionine

How many stop codons are there?

Three codons

What are synonyms for stop codons?

1. Nonsense codons

2. Termination codons

Why are some codons “stop” codons?

No tRNA corresponds to them, thus no amino acid is encoded by them

Is the genetic code universal?

Mostly!

Some Exceptions

Define translation

The process by which ribosomes synthesize proteins is by decoding the messenger RNA (mRNA) sequence into a corresponding amino acid sequence, facilitating the assembly of polypeptide chains.

Where does translation occur within the cell?

In the cytoplasm, specifically on ribosomes.

What is tRNA charging?

The process that allows the tRNA to be loaded (charged) with the appropriate amino acid

What are the 4 steps of translation

1. tRNA charging

2. Initiation

3. Elongation

4. Termination

Explain the process of translation initiation in prokaryotes as detailed below

1. IF3 binds to a small ribosomal subunit and prevents the large subunit from binding during initiation

2. IF3 helps bring the small subunit complex to the mRNA, where the Shine-Dalgarno sequence is within the mRNA.

3. The rRNA in the small subunit is complementary to the Shine-Dalgarno sequence

4. A tRNA (carrying the anticodon) charged with N-formylmethionine forms a complex with IF-2 and GTP, which then binds to the start (initiation) codon

5. IF-1 joins the small subunit once the anticodon is bound

6. GTP is hydrolyzed to GDP and all of the IF’s leave the complex

7. Large ribosomal subunit joins

Explain the process of translation elongation in prokaryotes

1. After initiation, the fMET-tRNA is in the P site of the ribosome.

2. EF-Tu (Elongation factor-Tu), GTP, and charged tRNA formed a complex that enters the A site of the ribosome

3. After the charged tRNA is placed in the A site, GTP is hydrolyzed and cleaved to GDP. Then, the EF-TU/GDP complex is released

4. ***EF-Ts regenerate the EF-Tu/GTP complex, which is then ready to combine with another charged tRNA

5. A peptide bond is formed between the amino acids in the P and A sites, (peptidyl transferase is an enzyme that does this)

6. tRNA is in the P site, and it releases its amino acid, so now the peptide chain is entirely on the tRNA at the A site

7. The ribosome shifts down by one codon (translocation) with the help of EF-G and GTP

8. The tRNA that was in the P site is now in the E site, from which it immediately leaves and moves into the cytoplasm

9. The A site is now available to receive the next charged tRNA and the cycle continues. The amino acid in the P site is now in the E site and then it immediately leaves

10. The A site is now available to receive the next charged tRNA and the cycle continues

Explain the process of translation termination in prokaryotes

1. The ribosome translocates to a stop codon, and when the stop codon is in the A site, there is no tRNA with an anticodon that can pair

2. A release factor (RF-1) attaches to the A site

3. Another release factor (RF-3) joins the ribosome (large subunit) and forms a complex with GTP, so that everything is released from the tRNA, including the polypeptide sitting in the P site

4. GTP associated with RF-3 is hydrolyzed to GDP, and the tRNA, mRNA, and RF’s are released from the ribosome

Differentiate between translation initiation in prokaryotes and that of eukaryotes

Eukaryotic Transcription:

-The small subunit binds to the 5' cap of mRNA and scans the mRNA looking for AUG

-A complex assembly involves additional initiation factors, and the large ribosomal subunit joins after the complex is formed.

-The PolyA tail aids in this binding by looping around to the proteins bound to the 5’ cap, enhancing the binding of the small subunit to the 5’ end of the mRNA

-Translation initiation involves more initiation factors, and the initiator tRNA carries methionine

Prokaryotic Transcription:

-The small subunit binds to the Shine-Dalgarno sequence, and initiator tRNA carries methionine (not N-formylmethionine).

Explain simultaneous transcription and translation, and state whether this occurs in prokaryotic or eukaryotic cells

-In prokaryotic cells, transcription and translation occur simultaneously because there is no nuclear membrane separating the processes.

-As soon as mRNA is synthesized by RNA polymerase, ribosomes attach to it and begin translating it into protein while transcription is still ongoing.

-Polyribosomes are formed as multiple ribosomes translate one mRNA strand at the same time, increasing the efficiency of protein synthesis. They simultaneously translate mRNA molecules. Each ribosome attaches to the ribosome binding site initiating translation while the preceding polypeptide strand is being synthesized.

-POLYRIBOSOMES: occur in both prokaryotic and eukaryotic cells

-Eukaryotes cannot do this simultaneously because transcription occurs in the nucleus and translation occurs in the cytoplasm

Explain polyribosomes and state whether these occur in prokaryotic or eukaryotic cells

-Polyribosomes are complexes formed when multiple ribosomes attach to a single mRNA strand, allowing for simultaneous translation of the mRNA into protein.

-They occur in both prokaryotic and eukaryotic cells, but the process of simultaneous transcription and translation is primarily characteristic of prokaryotes due to the lack of a nuclear membrane.

-This arrangement increases the efficiency of protein synthesis by enabling many ribosomes to translate the same mRNA strand concurrently.

Define post-translational modifications and explain its importance

-Post-translational modifications are chemical changes (addition of chemical groups) to a protein that occur after translation, such as phosphorylation, glycosylation, and ubiquitination.

-The function of many proteins depends critically on the proper folding of the polypeptide chain

-Folding, trimming, and removal of signal sequence are essential for protein functionality and play a key role in modulating cellular processes, affecting how proteins interact with other molecules.

-Some proteins spontaneously fold into their correct shapes, but for others, correct folding may require molecular chaperones to fold

-These modifications are crucial for regulating protein activity, stability, localization, and interactions, ultimately influencing cellular functions.

Define “reading frame” as it applies to the genetic code

This refers to how nucleotides in a nucleic acid molecule are read as codons. Each sequence of nucleotides in an mRNA has three possible ones of these. This can be shifted by insertions or deletions of nucleotides, which can result in significant changes in the resulting protein sequence.

Define “overlapping code” as it applies to the genetic code

This refers to a scenario in which the same nucleotide sequence can be read in multiple ways, with the same nucleotides contributing to more than one codon. This means that different proteins can be produced from the same segment of DNA or RNA sequences, the same segment of the genome encodes for multiple polypeptides.

Define “nonoverlapping code” as it applies to the genetic code

This refers to a coding system where every single nucleotide in a DNA or RNA sequence is part of only one codon, meaning that each set of three nucleotides corresponds to a specific amino acid without sharing or overlapping with any other codons. This results in the production of a single type of polypeptide from one polynucleotide sequence.

Define “initiation codon” as it applies to the genetic code

This refers to a specific set of nucleotides that establishes the appropriate reading frame and specifies the first amino acid of the protein chain. Typically AUG. It marks the start of translation and codes for methionine in eukaryotes and N-formylmethionine in prokaryotes.

Define “termination codon” as it applies to the genetic code

This refers to a set of nucleotides that signals the end of translation and the end of the polypeptide molecule. There are three of them- UAA, UAG, UGA- which can also be referred to as stop codons or nonsense codons. Typically do not encode for amino acids

Define “sense codon” as it applies to the genetic code

This refers to a set of nucleotides that code for an amino acid. In a standard genetic code, there are 61 of these that code for the 20 amino acids commonly found in proteins.

Define “nonsense codon” as it applies to the genetic code

This refers to a type of termination codon that does not code for any amino acid and signals the termination of protein synthesis. Include UAA, UAG, and UGA.

Define “universal code” as it applies to the genetic code

This refers to the fact that each codon codes for the same amino acid in all organisms. This is universal with a few exceptions which are in mitochondrial genes.

Define “nonuniversal codons” as it applies to the genetic code

Refers to sets of nucleotides are not universally the same across all organisms, differing in certain mitochondrial and protist genes. A codon may have a different meaning in different organisms or organelle genomes.

How is the reading frame of a nucleotide sequence set?

The initiation codon, typically the first AUG on the mRNA sets this

What role do initiation factors play in protein synthesis?

They are required for the initiation of translation. Bacteria have three (IF-1, IF-2, and IF-3). IF-3: comes in first and binds to the small ribosomal subunit to prevent it from reassociating with the large subunit, thus allowing the small subunit to attach to the mRNA

• IF-2: forms a complex with GTP, then binds to the initiator tRNA, escorts the tRNA that is charged with the N-formylmethionine (fMet-tRNA) to the initiation codon on the mRNA, facilitating the formation of the initiation complex.

• IF-1: joins the complex with fMet-tRNA and GTP and IF-2, specifically on the small subunit of the ribosome. Promotes the separation of the large and small ribosomal subunits

  ***In eukaryotes, there are more IF’s with similar roles, and some of them are necessary for recognition of the 5’cap on the mRNA. Other IF’s have RNA helicase activity which help fix the secondary structures that may form on the 5’ UTR of the mRNA

What do each of the prokaryotic initiation factors do?

• IF-3: comes in first and binds to the small ribosomal subunit to prevent it from reassociating with the large subunit, thus allowing the small subunit to attach to the mRNA

• IF-2: forms a complex with GTP, then binds to the initiator tRNA, escorts the tRNA that is charged with the N-formylmethionine (fMet-tRNA) to the initiation codon on the mRNA, facilitating the formation of the initiation complex.

• IF-1: joins the complex with fMet-tRNA and GTP and IF-2, specifically on the small subunit of the ribosome. Promotes the separation of the large and small ribosomal subunits

How does the process of initiation differ in bacterial and eukaryotic cells

• Bacterial initiation of translation requires that sequences in the 16S rRNA of the small ribosomal subunit bind to the mRNA at the ribosome binding site (the Shine–Dalgarno sequence). The Shine–Dalgarno sequence is essential in placing the ribosome over the start codon (typically AUG).

• In eukaryotes, there is no Shine–Dalgarno sequence. The small ribosomal subunit recognizes the 5′ cap of the eukaryotic mRNA with the assistance of initiation factors. In addition, proteins that attach to the poly(A) tail interact with proteins that bind to the 5′ cap to enhance the binding of the small ribosomal subunit to the 5′ end of the mRNA.

• Next, the ribosomal small subunit migrates along the mRNA scanning for the AUG start codon. In eukaryotes, the start codon is located within a consensus sequence called the Kozak sequence (5′–ACCAUGG–3′). Translation in eukaryotes also requires more initiation factors.

Give the elongation factors used in bacterial translation, and explain the role played by each factor in translation.

Three EF’s

1. EF-TU: Joins with GTP and then attaches to a charged tRNA. This complex then enters the A site of the ribosome and the tRNA is delivered with the amino acid it has. GTP is hydrolyzed and EF-Tu and GDP form a complex which is released.

2. EF-Ts: Regenerates the EF-Tu-GTP complex after hydrolysis of GTP

3. EF-G: Binds GTP and facilitates the translocation of the ribosome along the mRNA, moving the tRNA in the A site to the P site. This is crucial for allowing the next charged tRNA to enter the A site and continue the elongation process.

What events bring about the termination of translation?

• Process of termination begins when a stop codon enters the A site. This stop codon has no corresponding anticodon/tRNA to bind to.

• This allows release factors to bind to the ribosome. In bacteria, RF-1 recognizes the stop codon and attaches to the A site, while RF-2 can recognize additional stop codons.

• RF-3—GTP forms a complex and binds to the large subunit of the ribosome.

• The polypeptide chain is released from the tRNA in the P site

• GTP is hydrolyzed, and the release factors, tRNA, and mRNA are all released from the ribosome.

• **In eukaryotes, there are only two release factors- eRF-1, which recognizes all stop codons, and eRF-2, which binds to GTP and stimulates the release of the polypeptide from the ribosome. Then the polypeptide chain is released from the tRNA in the P site and GTP is hydrolyzed to GDP

Compare and contrast the process of protein synthesis in bacterial and eukaryotic cells, giving similarities and differences in the process of translation in these two types of cells

Similarities:

1. Bacteria and eukaryotes both share the universal genetic code

2. tRNA is charged the same way in both

3. Both have large and small ribosomal subunits

4. In both, mRNAs are translated multiple times and are simultaneously attached to several ribosomes, forming polyribosomes

Differences:

1. However, the start codon (AUG) in eukaryotes codes for methionine, whereas in bacteria it codes for N-formylmethionine

2. Bacterial cells have simultaneous translation and transcription, while in eukaryotes, transcription takes place in the nucleus and translation occurs in the cytoplasm

3. Bacterial mRNA lack a 5’cap and poly A tail, and are short lived. Eukaryotic mRNA last longer.

4. Bacterial translation has an initiator tRNA that carries N-formylmethionine, and eukaryotic translation has an initiation tRNA that carries methionine

5. Bacterial large subunit is smaller than the eukaryotic large ribosomal subunit (two rRNAs and 52 proteins vs three rRNAs and 82 proteins)

6. During bacterial translation, the small ribosomal subunit attaches directly to the region of the mRNA surrounding the start codon via the Shine-Dalgarno sequence in the 5’ UTR of the mRNA. In eukaryotic mRNAs, the small subunit binds to the 5’cap and scans until it finds the first AUG codon.

7. More IF’s are involved in eukaryotic translation than bacterial translation