Cell and Molec Lecture 8

How many possible combinations are there with three-base codons?

64

What is the third position of a codon called?

The wobble position

What is a cell lysate?

A mixture containing cellular components released when cells are broken open, allowing biochemical reactions such as protein synthesis to occur.

Which codon only varies at the wobble position?

Proline

What is the important poly-U experiment?

Cell lysates were given synthetic RNA made entirely of uracil (poly-U), which caused the synthesis of polypeptides made entirely of phenylalanine.

What did the poly-U experiment demonstrate?

The UUU codon codes for phenylalanine.

How many codons encode methionine?

One: AUG

Do mRNA transcripts begin with the start codon?

No. There is usually untranslated sequence before the start codon.

What is the 5′ untranslated region?

The region of an mRNA located before the start codon that is not translated into protein.

What does UTR stand for?

Untranslated region

Where is the 5′ UTR located?

Before the start codon toward the 5′ end of the mRNA

What are the three stop codons?

UAA, UAG, and UGA.

What is the 3′ UTR?

The portion of mRNA after the stop codon that is not translated into protein.

What important sequence/structure is found beyond the stop codon in the 3′ region of mRNA?

The 3′ UTR, which includes the poly-A tail.

What do release factors do when they recognize a stop codon?

They alter the activity of the ribosome and stimulate the release of the newly synthesized peptide.

What is frame 1?

Translation begins with the first nucleotide of the sequence.

What is frame 2?

Translation begins with the second nucleotide of the sequence.

What is frame 3?

Translation begins with the third nucleotide of the sequence.

What establishes the correct reading frame?

The AUG start codon establishes the correct reading frame.

What are alternative start codons?

Non-AUG codons that can occasionally function as translation initiation sites.

What is an example of a gene that can use an alternative non-AUG start codon?

c-Myc

What molecule decodes mRNA codons and links RNA sequences to amino acid sequences?

Transfer RNA (tRNA)

What is the classic two-dimensional representation of tRNA called?

The cloverleaf or cruciform structure

What is the three-dimensional structure of tRNA?

It is roughly shaped like a letter L.

Why does tRNA have an L-shaped structure?

Its loops fold over one another, producing a three-dimensional L-shaped molecule.

What is the acceptor stem of tRNA?

The region where the amino acid is attached.

Where is the amino acid attached to tRNA?

At the 3′ acceptor stem/end of the tRNA.

What is the anticodon?

A three-nucleotide sequence in tRNA that pairs with a complementary codon in mRNA.

Why don't bacteria need 64 different types of tRNA?

Some tRNAs can recognize multiple codons that specify the same amino acid because of flexibility in base pairing.

Do mammalian cells have more types of tRNA than bacteria?

Yes

Where are many tRNA nucleotides modified?

In the nucleus.

What is inosine?

A non-standard, modified nucleotide that can make base pairing more flexible.

Where is inosine especially important?

At the wobble position of tRNA anticodons.

In eukaryotes, what bases can inosine pair with at the wobble position?

U or C

In bacteria, what bases can inosine pair with at the wobble position?

U,C or , A

In which direction is mRNA normally read during translation?

5’ —→ 3’

Which position of the anticodon corresponds to the wobble position

The first nucleotide of the anticodon.

What enzymes attach amino acids to the correct tRNAs?

Aminoacyl-tRNA synthetases.

What is the main function of aminoacyl-tRNA synthetases?

They attach the correct amino acid to its corresponding tRNA.

What provides the energy required for amino acid attachment to tRNA?

ATP hydrolysis

What is the activated intermediate in amino acid attachment to tRNA?

An adenylated amino acid, in which AMP has been conjugated to the amino acid.

What nucleotide is attached to the amino acid during activation?

AMP

How do aminoacyl-tRNA synthetases ensure the correct amino acid is attached to a tRNA?

They have specific binding sites for the correct amino acid and corresponding tRNA.

Can an incorrect amino acid sometimes fit into the initial binding pocket?

Yes

What is the editing pocket of an aminoacyl-tRNA synthetase?

A proofreading site where incorrectly activated amino acids can be hydrolyzed and removed.

Why is proofreading by aminoacyl-tRNA synthetases important?

It prevents incorrect amino acids from being attached to tRNAs and ultimately incorporated into proteins.

How accurate are aminoacyl-tRNA synthetases

They make mistakes approximately once in every 40,000 couplings.

How can bacteria function with fewer than 20 aminoacyl-tRNA synthetases?

Other enzymes can chemically modify amino acids after they have been attached to tRNAs so that the tRNAs correctly correspond to the appropriate anticodons.

What is the first adapter in genetic-code decoding?

Aminoacyl-tRNA synthetases

What does the first adapter in genetic-code decoding do?

It correctly matches an amino acid with the appropriate tRNA.

What is the second adapter in genetic-code decoding?

tRNA

What does the tRNA do as the second adapter in gentic-code decoding?

It uses its anticodon to recognize and bind the appropriate codon on mRNA.

What is "tRNA charging"?

The process of attaching an amino acid to its corresponding tRNA.

What is the overall pathway connecting an mRNA codon to the correct amino acid?

Aminoacyl-tRNA synthetase correctly attaches an amino acid to a tRNA → the tRNA's anticodon recognizes the appropriate mRNA codon → the amino acid is incorporated into the growing polypeptide.

What is the fundamental function of ribosomes?

They recruit tRNAs to mRNA in a sequential and systematic manner, allowing codon-anticodon pairing and catalyzing peptide bond formation.

In what direction does the ribosome move along mRNA?

5’—→3’

What do tRNAs do during translation?

They bring amino acids to the ribosome and use their anticodons to base pair with complementary codons on the mRNA.

What catalytic activity does the ribosome have that forms peptide bonds?

Peptidyl transferase activity

What is the direction of polypeptide synthesis?

N-terminus → C-terminus

What is a peptidyl-tRNA?

A tRNA that is covalently attached to the growing polypeptide chain.

During translation, does the growing polypeptide remain attached to a tRNA?

Yes

What is a peptidyl-tRNA linkage?

The covalent linkage connecting the growing polypeptide to its tRNA.

What happens to the growing peptide when a new amino acid forms a peptide bond with it?

The peptide is transferred from the tRNA in the P site to the tRNA in the A site.

Why is peptide bond formation energetically favorable?

The growing C-terminus has been activated through its covalent attachment to a tRNA.

What is the bacterial ribosome called based on its sedimentation coefficient?

70S ribosome

What is the eukaryotic ribosome called based on its sedimentation coefficient?

80S ribosome

What is the bacterial small ribosomal subunit?

30S

What is the bacterial large ribosomal subunit?

50S

What is the eukaryotic small ribosomal subunit?

40S

What is the eukaryotic large ribosomal subunit?

60S

Approximately how large is the bacterial 30S small subunit?

About 900 kDa

Approximately how large is the eukaryotic 40S small subunit?

About 1,400 kDa.

Approximately how large is the bacterial 50S large subunit?

About 1,600 kDa

Approximately how large is the eukaryotic 60S large subunit?

About 2,800 kDa

What is 16S rRNA?

Ribosomal RNA that is part of the bacterial small 30S ribosomal subunit.

What important role does 16S rRNA play during translation?

It helps check whether the codon and anticodon are correctly base paired.

What are the three tRNA-binding sites in a ribosome?

A, P, and E sites

What does the A site stand for?

Aminoacyl site

What does the P site stand for?

Peptidyl site

What does the E site stand for?

Exit site

In what order do tRNAs move through the ribosome?

A → P → E

What happens at the A site?

Incoming aminoacyl-tRNAs enter and are checked for correct codon-anticodon pairing.

What happens at the P site?

The tRNA carrying the growing polypeptide is positioned here.

What happens at the E site?

The empty tRNA exits the ribosome.

How many tRNAs can the ribosome accommodate at one time?

Two

Why is the close positioning of the A and P sites important?

It ensures that codons are read sequentially and that no codons or individual bases are accidentally skipped.

What primarily forms the A, P, and E sites?

Ribosomal RNA

What is present in the P site at the beginning of an elongation cycle?

A tRNA carrying the growing polypeptide.

What is present in the A site at the beginning of an elongation cycle?

A newly arrived aminoacyl-tRNA carrying the next amino acid.

What happens during peptide bond formation in the elongation cycle?

A peptide bond forms between the existing polypeptide and the amino acid attached to the tRNA in the A site.

What happens to the growing polypeptide after peptide bond formation?

It is transferred from the tRNA in the P site to the tRNA in the A site.

What does translocation mean during translation?

Movement of the ribosome along the mRNA by exactly one codon.

In which direction does the ribosome translocate along the mRNA?

5′ → 3′

What happens to the tRNAs during translocation?

They temporarily occupy hybrid sites between the large and small subunits.

What are elongation factors?

Proteins that regulate and help drive the translation elongation process.

What are the two major elongation factors?

EF-Tu and EF-G in bacteria

What is the eukaryotic equivalent of bacterial EF-Tu?

EF1

What is the eukaryotic equivalent of bacterial EF-G?

EF2

What is the main function of EF-Tu?

It delivers incoming tRNAs to the A site and helps increase the accuracy of translation.

What nucleotide does EF-Tu bind?

GTP