Transcription and Translation

7.3 Transcription

Nucleosides vs. Nucleotides

•         A nucleoside: has a nitrogen base linked by a glycosidic bond to C1’ of a sugar (ribose or deoxyribose) (without the phosphate group)
•         A nucleotide is a nucleoside that forms a phosphate ester with the C5’ –OH group of a sugar (ribose or deoxyribose).
•         Phosphodiester bond join nucleotides

       Nucleoside                                                                         Nucleotide

                                                        

Types of RNA

•       Messenger RNA (mRNA)- carries genetic information from DNA to cytosol

•       Ribosomal RNA (rRNA)- Most abundant; makes up ribosomes

•       Transfer RNA (tRNA)- Binds to specific Amino Acids

RNA

•       Transcription- the copying of the base sequence of a gene (DNA) by making an RNA molecule.

•       Complementary base pairing rules are followed CºG except that A=U

Transcription

•         5’ end of free nucleotides are added to the 3’ end
•        The sense strand (coding strand) has the same base sequence as mRNA with uracil instead of thymine. – 5’ to 3’
•        The antisense (template) strand is transcribed

Transcription Process

Initiation Explained

•       The promoter region is for the binding of RNA polymerase. 

•       This allows RNA Polymerase to:

•       Find the Anti-sense strand, know the direction of transcription, and the start for transcription.

•       The hydrogen bonds of the DNA helix are opened by the DNA Helicase.

•       The bases of the anti-sense strand (3’-5’ for DNA) are exposed.

•       RNA Nucleotides complementary base pair with the anti-sense nucleotide bases

•       The free nucleotides (nucleoside triphosphates) are based on RNA.  The sugar is Ribose.

•       The nucleotides are Adenine, Guanine, Cytosine and Uracil.    

Elongation Explained

•         The RNA polymerase forms covalent bonds between nucleotides (phosphodiester).
•         Free energy is released from the oxidation reaction of the nucleoside triphosphates to form the bond.
•      The bonds are formed by joining the 5’ of the free nucleotide to the 3’ end of the nucleotide already part of the mRNA chain
•      The RNA polymerase works along the nucleotides completing the ribose-phosphate backbone

Steps of Transcription

1. Helicase uncoils the DNA by breaking the hydrogen bonds of the complementary base pairs at the position of the gene.
2. RNA polymerase finds the promoter region on the antisense strand of DNA (TAC)- Initiation.
3. Free RNA nucleotides complementary base pair with DNA nucleotides on the antisense strand. A=U, GºC
4. The phosphodiester bonds on the mRNA chain are formed by RNA Polymerase- Elongation.
5. The RNA polymerase reaches the terminator (DNA- ATT, ATC, or ACT) and the RNA polymerase stops.
6. The mRNA is complete the molecule detaches from the DNA and leaves the nucleus for the cytoplasm ribosomes.
7. The DNA helix reforms

SNRPS

•       Are used to remove introns to form mature mRNA

•       Pre-mRNA has been produced through transcription of the anti-sense strand as described for prokaryotic transcription.

•       (a) The non-coding introns are spliced out of the mRNA.

•       The introns are broken down in the nucleus.

•       (b) The remaining mRNA is called mature mRNA and is exported from the nucleus to the cytoplasm for translation into the polypeptide.

7.4 Translation

One Gene, One Polypeptide

•       Theory- One gene is transcribed and translated to produce one polypeptide.

•       Some proteins are composed of a number of polypeptides and in this theory each polypeptide has its own gene.

•       e.g. Hemoglobin is composed of 4 polypeptides (2 of each type) and there is a gene for each type of. polypeptide.

Codons

•       Genetic code- A T(U) G C, is used by most organisms to translate mRNA into proteins.

•       Codon- 3 (triplet) nucleotides of mRNA that code for an amino acid.

•       Anticodon- 3 nucleotides of tRNA that are complementary to and pairs with the mRNA codon. They carry the amino acid.

•       A polypeptide is a sequence of bases

•       Bases are either A,T,G, or C

•       64 codons code for 20 amino acids.

•       Less of a chance for mutations

•       The genetic code is universal so it is the same in almost all organisms

•       Each codes for the addition of an amino acid to a growing polypeptide chain

•       The genetic code is degenerate- meaning more than one codon can code for a particular amino acid

•       AUG is the start codon

•       Some codons code for the end of translation- Terminator codons (UGA, UAA, UAG)

Translation

•       The location of translation is the ribosomes in the cytoplasm.

•       Ribosomes are composed of rRNA which acts as a catalyst for the translation of mRNA.

•       mRNA from the nucleus locates a ribosome.

•       The start codon (AUG) occupies one of two ribosome sites.

•       The ribosome moves along the mRNA

•       One mRNA can have many ribosomes (polysome) which accelerate protein synthesis.

tRNA

•  Each amino acid has a specific tRNA-activating enzyme (aminoacyl-tRNA synthetase)

•       tRNA is composed of one chain of (RNA) nucleotides

•       tRNA has an anticodon

•       Anticodon of three bases which are single stranded and form part of a loop

•       tRNA has double stranded sections formed by base pairing

•       tRNA has 3 loops (sometimes with an extra small loop)

•       tRNA has a distinctive 3D clover leaf shape

•       Activation specificity: how does the tRNA attach to the correct amino acid.

•       Shape of each tRNA is different.

•       Shape of the tRNA is defined by the loop and the helical sections.

•       The enzyme adds a specific amino acid to the CCA base sequence (at 3' end of the tRNA) this requires ATP (energy).

•       Each amino acid has one or more tRNA molecules- example of a degenerate code.

Binding Sites for tRNA

•       A- Amino Acid- is the position that the new tRNA codon-anticodon binds making sure that the correct amino acid is in position.

•       P- Polypeptide- is the position in which the amino acid on the tRNA adds to the polypeptide.

•       E- Exit- is the position the tRNA (w/o amino acid) locates and is then released from the ribosome to become reactivated.

Translation Explanations

•       Initiation: In which the ribosome, tRNA and mRNA come together to begin the translation of the mRNA.

•       Elongation: tRNA molecules attach to the mRNA based on the codon-anticodon recognition. Amino acids are brought together and polymerized into the primary structure of the polypeptide.

•       Translocation: The movement of the ribosome along the mRNA strand one codon at a time.

•       Termination: mRNA and the ribosomes detach from one another. The polypeptide is released and the tRNA return to be charged with more amino acid.

Steps of Translation

1. Initiation- ribosomes bind to mRNA.  Initiator tRNA binds the start codon to the small subunit of ribosome.
2. The start codon (AUG- methionine) occupies the P site.
3. Elongation- ribosomes moves along mRNA facilitating addition of amino acids.
4. Second tRNA binds to ribosome at the A site.
5. Large subunit moves down mRNA after a second tRNA binds.
6. The amino acid on first tRNA is bonded to amino acid on second tRNA.
7. Peptide bonds between amino acids are formed with the aid of peptidyl transferase.  Requires GTP- Guanosine-5'-triphosphate.
8. The ribosome translocates- moves down the mRNA.
9. The first tRNA moves to the E site and is removed and new tRNA binds.
10. Each tRNA moves from the A site to the P site to the E site.
11. Termination- the ribosome reaches a stop codon The stop codon causes the polypeptide to be released. 

Ribosomes

•        free ribosomes synthesize proteins for use primarily within the cell
•        bound ribosomes synthesize proteins primarily for secretion by lysosomes
•     Proteins and Ribosomal RNA combine in the structure.
•        Large sub-unit has three binding sites for tRNA molecules (E, P and A site).
•        Small sub-unit has a binding site for mRNA.
•        Ribosome Function: Ribosomes are enzymes that catalyze the translation of mRNA into a polypeptide- their substrate is mRNA

Peptide Bond

                           

Comparing Transcription and Translation