Lecture 8: DNA replication, transcription and translation

Question 1

DNA replication

Answer 1

begins at Origin of replication
two strands open forming replication forks ( y-shaped region)
new strands grow at the forks

as the 2 DNA strands open at the origin replication bubbles form
-prokaryotes have a single bubble
-eukaryotic chromosomes have many bubbles

Enzyme Helicase unwinds and separates the 2 DNA strands by breaking the weak hydrogen bonds
Single-Strand Binding Proteins attach and keep the 2 DNA strands separated and untwisted

Enzyme Topoisomerase attaches to the 2 forks of the bubble to relieve stress on the DNA molecule as it separates

Before new DNA strands can form, there must be RNA primers present to start the addition of new nucleotides
Primase is the enzyme that synthesizes the RNA Primer

DNA polymerase can only add nucleotides to the 3’ end of the DNA
This causes the NEW strand to be built in a 5’ to 3’ direction

Question 2

Mechanism of DNA polymerase

Answer 2

DNA polymerase can then add the new nucleotides

Question 3

DNA polymerase can only add the new nucleotides 5’ to 3’- DNA replication

Answer 3

But the two strands of DNA are in opposite directions.

So we have a leading strand and a lagging strand.

The Leading Strand is synthesized as a single strand from the point of origin toward the opening replication fork

The Lagging Strand is synthesized discontinuously against overall direction of replication
This strand is made in MANY short segments It is replicated from the replication fork toward the origin

Question 4

Lagging strand fragments

Answer 4

Okazaki Fragments - series of short segments on the lagging strand
Must be joined together by an enzyme

The enzyme DNA Ligase joins the Okazaki fragments together to make one strand

Question 5

Semi-conservative model of replication

Answer 5

The two strands of the parental molecule separate, and each acts as a template for a new complementary strand
New DNA consists of 1 PARENTAL (original) and 1 NEW strand of DNA

Question 6

Transcription : An overview

Answer 6

During transcription the mRNA molecule is synthesised by a process similar to DNA replication except that :

the enzyme is RNA polymerase
RNA polymerase does not require a primer
RNA polymerase does not 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 (not thymine)
unlike DNA replication, mRNA synthesis starts at many places on the DNA
sequence and stops at predetermined places.
unlike DNA synthesis, when synthesis of a mRNA molecule is complete, it is
released from the template as a single stranded molecule

Note that like DNA synthesis, RNA synthesis takes place in a 5’ to 3’ direction

Question 7

Transcription : Start Signal in Prokaryotes

Answer 7

The principal new information that is required to understand transcription is knowing where the process starts. The DNA sequence at which mRNA synthesis starts is determined by a section of the DNA called a promoter.

For every section of DNA coding for a protein there has to be a promoter, although in some cases two or more genes share one promoter.

If one or more genes, usually with related functions, share a promoter, this group of genes are described as an operon

In E.coli the promoter sequences for a wide range of genes have been shown to consist of two characteristic sequences separated by about 25 nucleotides

Question 8

Translation : An overview

Answer 8

Translation is the second major step in protein synthesis. It involves using the information carried on the mRNA to join amino acids together in the sequence required to produce a specific protein.

The information on the mRNA is carried by the sequence of bases arranged in a triplet code represented in the table

Each triplet is called a codon

For example, CAC will be translated as the amino acid histidine, whereas CAG will be translated as glutamine

Three triplets, UAA, UAG and UGA do not code for amino acids and are called stop or nonsense codons. These serve as stop signals for translation

Question 9

Translation : The triplet code

Answer 9

It is degenerate, that is some amino acids are coded for by more than one codon. UUU and UUC both code for phenylalanine.

AUG is unique in that it codes unambiguously for methionine. It is also important as always being the first codon that is translated on mRNA. It is the start signal for translation.

The triplet code is universal in all organisms with only a few exceptions (below).

Question 10

Translation : The process

Answer 10

Steps of Translation:

1. Initiation:
   
   • The small ribosomal subunit binds to the mRNA near the 5’ cap in eukaryotes or the Shine-Dalgarno sequence in prokaryotes.
   • The initiator tRNA, carrying the amino acid methionine (Met), binds to the start codon (AUG) on the mRNA.
   • The large ribosomal subunit then joins to form the complete ribosome, positioning the initiator tRNA in the P site.
2. Elongation:
   
   • The next codon on the mRNA is recognized by the corresponding aminoacyl-tRNA, which binds to the A site of the ribosome.
   • A peptide bond is formed between the amino acid in the P site and the amino acid in the A site. This reaction is catalyzed by the ribosomal RNA (rRNA) in the ribosome.
   • The ribosome then moves one codon down the mRNA (translocation), shifting the tRNA with the growing polypeptide to the P site and the empty tRNA to the E site, from which it exits the ribosome.
   • The A site is now ready to accept a new aminoacyl-tRNA corresponding to the next codon.
3. Termination:
   
   • When a stop codon (UAA, UAG, or UGA) is encountered on the mRNA, release factors bind to the A site.
   • These factors promote the release of the polypeptide chain from the tRNA in the P site.
   • The ribosome disassembles into its subunits, releasing the mRNA and the newly synthesized protein.

Key Points:

• Ribosome Structure: Ribosomes are composed of a small and a large subunit, which contain ribosomal RNA (rRNA) and proteins.
• tRNA Role: Transfer RNA (tRNA) molecules have an anticodon region that is complementary to the mRNA codon and an attached specific amino acid.
• Codon-Anticodon Interaction: The mRNA codons are read in the 5’ to 3’ direction, and each codon specifies an amino acid according to the genetic code.

Regulation and Efficiency:

• Initiation Factors: Proteins that assist in the assembly of the ribosome and the initiator tRNA on the mRNA.
• Elongation Factors: Proteins that facilitate the binding of tRNA to the ribosome and translocation of the ribosome along the mRNA.
• Termination Factors: Proteins that recognize stop codons and trigger the release of the newly synthesized protein.

Translation is a crucial process in gene expression, converting the genetic code into functional proteins that perform various roles in the cell.