Lecture 8 - DNA replication, transcription and translation

Question 1

Where does DNA replication begin along the DNA molecule?

Answer 1

At origins of replication.

Question 2

Describe this image.

Answer 2

The double helix is broken apart and two polynucleotide strands open forming replication forks. New strands grow at forks- they read along from the origin of replication.
DNA is copied at many points along the DNA molecule. As DNA strands open along the DNA molecule, replication bubbles form. The bubbles eventually get bigger, join and we have identical copies of both DNA strands.

Question 3

Role of enzyme helicase in DNA replication.

Answer 3

Enzyme helicase unwinds and separates the two strands by breaking the hydrogen bonds between base pairs

Question 4

Role of single strand binding proteins in DNA replication

Answer 4

Single-strand binding proteins attach to each strand and keep the two DNA strands separated and untwisted.

Question 5

Role of topoisomerase

Answer 5

Topoisomerase binds further along the DNA molecule. It attaches to the two forks of the bubble and its role is to relieve stress on the DNA molecule caused from the unwinding/separating of the double helix.

Question 6

Role of primase enzyme and primase.

Answer 6

RNA primers are synthesised by primase enzyme- the primers kick start the addition of new nucleotides

Question 7

Describe the work of DNA polymerase in DNA replication. Explain the direction in which the new strand is built and bonds that form in the process.

Answer 7

DNA polymerase moves along the strand and adds nucleotides to the 3’end of the DNA so the new strand is built in a 5’ to 3’ direction. Phosphodiester bonds form between the sugar and phosphates of the new nucleotides and hydrogen bonds from between complementary base pairs. Same process on repeat to form the polynucleotide strand.

Question 8

As we know, DNA polymerase only adds new nucleotides to the …… end of the template strand so the new strand is made in the …….-……… direction.

Answer 8

3’ end
5’-3’ direction

Question 9

The DNA polymerase can only add the new nucleotides to the 3’ end of the template strand so makes the new strand in the 5’ to 3’ direction, as we know.
But, we also know that the two polynucleotide strands in a DNA molecule are antiparallel. So, what is the effect?

Answer 9

In DNA replication we have a leading strand and a lagging strand.

Question 10

What is the leading strand?

Answer 10

The leading strand is synthesized as a single strand from the origin towards the opening of the replication fork.

Question 11

Describe the lagging strand.

Answer 11

The lagging strand is synthesized discontinuously- in small fragments (Okazaki fragments) against the overall direction of replication- from the replication fork towards the origin.

Question 12

What enzyme joins Okazaki fragments together to make one continuous strand?

Answer 12

DNA ligase.

Question 13

DNA must be proofread. Explain.

Answer 13

• DNA polymerase will make about 1 in 10,000 base paring errors.
• DNA polymerase can proofread and correct the mistakes. It can tell if something isn’t base paired correctly and will chop it off.

Question 14

What does semi conservative replication mean?

Answer 14

The two strands of the parental molecule separate and each strand acts as a template for a new complementary strand. New DNA consists of 1 parental/original and 1 new strand of DNA.

Question 15

DNA is not just used to store genetic info but also to synthesise proteins, therefore determining the cell structure and function. What is the definition of gene expression?

Answer 15

Gene expression is defined as the transformation of DNA information into functional molecules.

Question 16

Two stages of protein synthesis.

Answer 16

transcription and translation.

Question 17

What is transcription?

Answer 17

The production of a copy of a section of DNA carrying information related to one or more genes as a messenger mRNA molecule.

Question 18

How does transcription differ from RNA replication?

Answer 18

The enzyme is RNA polymerase
RNA polymerase does not require a primer
RNA polymerase does not need to proof read the synthesis
the sugars in the nucleic acid backbone are ribose not deoxyribose
the four bases used are adenine, guanine, cytosine and uracil
mRNA synthesis starts at many different small sections on the DNA sequence and stops at specific places
a single stranded mRNA molecule is produced

Question 19

In transcription of prokayrotes, there is a position along the DNA molecule at which transcription starts. What is the section of DNA called?

Answer 19

promoter region.

Question 20

Every gene- every section of DNA which codes for a protein- has a promoter region. What is it’s role? If one or more genes share a promoter what is it called?

Answer 20

The promoter directs the RNA polymerase to move along the strand in only one direction. If one or more genes share a promoter, this group of genes is described as an operon.

Question 21

Give the stages of transcription.

Answer 21

1. RNA polymerase binds to promoter sequence in duplex DNA- closed DNA complex
2. The RNA polymerase breaks apart the duplex DNA near transcription start site forming a transcription bubble- open complex
3. The RNA polymerase catalyses phosphodiester linkages of two initial ribonucleotides.
4. RNA polymerase moves along the template strand in the 3’-5’ direction, breaking apart the duplex DNA molecule and joining one ribonucleotide at a time to the growing RNA polynucleotide chain. We name this step elongation.
5. Termination: at the transcription stop site, polymerase releases and the complete RNA dissociates from DNA.

Question 22

What is translation?

Answer 22

The sequence of information on the mRNA molecule is used to synthesise a polypeptide strand- a sequence of amino acids

Question 23

Where does translation take place?

Answer 23

ribosomes

Question 24

During translation, three bases along the mRNA are read at a time. What do we call these three bases?

Answer 24

triplet/codon

Question 25

One triplet codes for…

Answer 25

one amino acid

Question 26

Three triplets that don’t code for amino acids are stop codons. Give the three

Answer 26

UAA, UAG, UGA

Question 27

What does degenerate code mean?

Answer 27

one amino acid may be coded for by multiple different triplets

Question 28

What is the start codon? It only codes for one amino acid- what is it?

Answer 28

AUG- methionine. It is the first codon that is translated on mRNA.

Question 29

What is the role of tRNA?

Answer 29

acts as the essential link between the code on the mRNA and the assembly of the amino acids into a polypeptide chain.

Question 30

All tRNA have same basic structure. What?

Answer 30

• fold into clover leaf shape
• contain an anticodon- bases complementary to codon on the mRNA.
• acceptor stem/site - where amino acids bind.

Question 31

Each tRNA has a different fine 3D structure. Why?

Answer 31

They must be specific to their appropriate aminoacyl- tRNA synthase.

Question 32

What is the role of aminoacyl-tRNA synthase?

Answer 32

tRNA molecules are covalently bonded to a specific amino acid. The correct amino acid is added to the tRNA that bears the correct anticodon. There is a specific aminoacyl-tRNA enzyme to link each tRNA with it’s appropriate amino acid.

Question 33

Peptide bonds form between the amino acids that are attached to the tRNA molecules. What is the enzyme responsible for peptide bond formation? draw the peptide bond formation.

Answer 33

peptidyl transferase.

Question 34

What are ribosomes in eukaryotes made up of?

Answer 34

rRNA and proteins.
each ribosome is made up of two subunits- 60s and 40s.

Question 35

Describe the steps of initiation in translation

Answer 35

1. translation starts at the first AUG codon closest to the 5’ end of the mRNA.
2. The initiator tRNA (methionine tRNA) binds to the small subunit of the ribosome.
3. The small subunit initiator tRNA complex binds to the mRNA and moves along until it locates the AUG start codon. The initiator tRNA has a complementary anticodon to this AUG start codon.
4. The initiator factors dissociate and the large rRNA subunit binds, enclosing the mRNA between the two subunits. There are two binding sites- P and A – on the ribosome. The second tRNA bearing the second amino acid binds to the A site (the met tRNA is on the P site)

Question 36

Describe elongation in translation.

Answer 36

5. Peptide bonds form between the amino acids attached to the tRNA in the P and A site molecules. The enzyme peptidyl transferase catalyses the reaction.
6. The first tRNA molecule leaves the P site and the ribosome complex moves along the mRNA molecule. The A site is now free for another tRNA molecule (the second tRNA linked to the peptide chain is moved along to the P site). Note: P site always holds the tRNA linked to the portion of the protein chain that has been assembled up to this point, and site A holds the tRNA containing the next amino acid to be added to the chain.
7. This process continues to form a long polypeptide chain made up of many amino acids joined via peptide bonds.

Question 37

Describe termination in translation.

Answer 37

8. The ribosome moving along the mRNA will eventually reach a stop codon (UAG, UAA, UGA). Instead of tRNA binding to the codon, a release factor binds in the A site.
9. There is no amino acid attached to the release factor and so nothing for peptidyl transferase to act on so the polypeptide chain is terminated.
10. The ribosomal subunits separate and the mRNA is released.