3.2 ~ DNA replication, transcription and translation

Separation of DNA

* DNA helicase unwinds and unzips DNA
* Separates the two polynucleotide strands by **breaking the H bonds** between complementary bas pairing
* **ATP** is needed by helicase to both move along the DNA molecule and to break the H bonds
* The two separated strands become parent/template strands for the replication process

Semi-conservative

* Means: half
* In this case : **conserved** or **saved**
* The two strands from the original DNA molecule remain in the new DNA molecules that are formed

Conservative

Means : whole

Dispersive

Means : separated randomly

DNA polymerase

* DNA polymerase synthesizes a new DNA strand in the 5’ to 3’ direction
* Enzymes
* Moves up to strand to create the new strand
* 5’ to 3’ refers to the particular location of one of the C atoms on the deoxyribose

Helicase

* Unwounds the DNA

Process of separation

1. DNA is unwound and strands and separated by helicase
2. The original strands are used as templates to copy
3. DNA polymerase is the enzyme that adds new nucleotides to the strands to finish creating two new DNA strands that are identical to the original
4. The matching of ‘A’ to ‘T’ and ‘C’ to ‘G’ is called complementary base pairing and this helps to conserve the base sequence of DNA
5. DNA replication is semi-conservative

What does semi-conservative mean

* In the end, when all is separated, it will be an exact copy of the original copy of the parent
* Two identical copies are created from the original copy from the original copy itself
* Each of the new double-helix strand contains one of the original ‘parent’ strands

Complimentary base pairing

Adenine is paired with thymine - Guanine is paired with cytosine

Semi-conservative

The two strands from the original DNA remain in the new DNA molecules formed. A new DNA double helix will contain one strand from the parents strand and one will contain one new strand

Intro to PCR

* Quick way to amplify minute quantities of DNA to obtain millions of copies of DNA
* DNA polymerase is used in PCR originated from the thermophilic microorganisms and therefore are heat resistant
* Ability to withstand high temp is key as high temp are needed to melt or separate the double stranded DNA
* Separation allows polymerase to synthesise complimentary DNA strands

Method for amplifying

1. Temp is raised to near boiling, causing the double-stranded DNA to separate, or denature, into single strand

2. When temp is decreased, short DNA sequences known as primer bind, or anneal, to complementary matches on the target DNA seqeunce

3. The primers bracket the target sequences to be copied

4. At a slightly higher temp, the enzyme TAQ polymerase, binds to the primed sequences and adds nucleotides to extend the second strand

   1. Completes the first process

5. In subsequent cycles, the process of denaturing, annexing and extending are repeated to make additional DNA copies

6. After 3 cycles, the target sequence defined by the primers begins to accumulate

7. After 30 cycles, as many as a billion copies of the target sequences are produced from a single starting molecules

To summarise

* PCR is a way of producing large quantities of a specific target sequence of DNA from a small sample
* It is useful when only a small amount of DNA is available for testing
* E.g. crime scene samples of blood, semen, hair, tissue
* One cycle of PCR yields two identical copies of the DNA sequence

Denaturation

DNA sample is heated to 95C which breaks the H bonding between the strands

Annealing

Same is called to 54C and mixed with DNA primers which attach to opposite ends of the target sequence

Elongation

At 72C, Taq DNA polymerase adds free DNA nucleotides to the strands of the DNA, using the primers as a starting point

Meselson and Stahl’s work

* Provides evidence for semi-conservative replication
* All bases are nitrogenous bases, meaning they contain N
* They are different isotopes of N
* Most are N14; lighter
* Some are N15; denser
* A centrifuge separates based on density
* Following centrifugation, denser molecules will be closer to the bottom

DNA bases

A-T

C-G

RNA bases

A-U

C-G

Transcription - general overview

* Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase
* Translation is the synthesis of polypeptides on ribosomes
* Three main types of RNA are predominantly synthesized
* Messenger RNA (mRNA): A transcript copy of a gene used to encode a polypeptide
* Transfer RNA (tRNA); A clover leaf shaped sequences that carries an amino acid
* Ribosomal RNA (rRNA); A primary component

Transcription process

• The enzyme RNA polymerase binds to a site on the DNA at the start of a gene

  • The sequence of DNA that is transcribed into RNA is called a gene

• RNA polymerase separates the DNA strands and synthesized a complementary RNA copy from the antisense strand

• DNA is unwound

• New nucleotides attach to template strand via complementary base pairing (A-U, C-G)

• Once the RNA sequence has been synthesized

  • RNA polymerase will detach from the DNA molecule

  • RNA detaches from the DNA

  • The double helix rewinds

• Transcription occur in the nucleus (where the DNA is) and, once made, the mRNA moves to the cytoplasm where translation can occur

Translation

* tRNA binds to ribosome at site where anticodon on tRNA corresponds to codon on mRNA
* mRNA molecule binds to ribosome (made of small and large subunit)
* Large subunit binds to tRNA molecule and catalysis peptide bonds between amino acids
* Small subunit of ribosome binds to mRNA molecule
* The first codon is called the start codon is called the start codon
* Codons of 3 bases on mRNA corresponds to one amino acid in a polypeptide
* Sequence of amino acids is determined by mRNA according of the genetic code
* Chain of amino acids
* Held together by peptide bonds

Translation process

* Ribosome moves along mRNA strand, reading 1 codon (3 nucleotides) at a time
* First tRNA molecule detaches
* Adjacent amino acids bind via peptide bonds
* Continues until stop codon reached
* Stop codon has no corresponding anticodon and thus no amino acid
* The stop codon signals that the polypeptide is complete

Summary of transcription and translation

* Translation is the process of protein synthesis in which genetic information encoded in mRNA is translated into sequences of amino acids in a polypeptide chain
* A ribosome is composed of 3 halves, a large chain and a small subunit
* During translation, ribosomal subunits assemble together like a sandwich on the strand of mRNA
* Each subunit is composed of RNA molecules and proteins
* The small subunit binds to the mRNA
* The large subunit has binding sites for tRNAs and also catalyses peptide bonds between amino acids

Sense and antisense

One side of the double helix is antisense and the other is the sense strand. The complementary RNA will be copied by RNA polymerase from the antisense DNA strand. This means that the RNA copy will be identical to the sense strand.

The Genetic code

* The genetic code is universal
* All living things use the same amino acid in transcription and translation, regardless of the species
* So, the sequence of amino acids in a polypeptide remains unchanged
* Therefore, we can take genes from one species and insert then into the genome of another species
* 64 codons but 20 amino acids