DNA and Genomics II (Transcription and Translation)

Why must DNA be transcribed onto mRNA?

DNA molecules are too large to move through nuclear pores in a nuclear envelope, but mRNA can pass through

What is transcription?

The process in which the nucleotide sequence of a gene is used as a template to direct synthesis of RNA made up of complementary base sequences

What does a gene consist of?

• Promoter → Non-coding DNA sequences which function as a recognition site for binding of general transcription factors and RNA polymerase to initiate transcription → Determine which strand to use as a template

• Transcription unit → Sequence of DNA flanked by the promoter and termination sequence → Transcribed into RNA

• Termination sequence → End of a gene that causes RNA synthesis to stop

What do the 2 strands of DNA function as in transcription?

• Template strand → Non-coding strand (mRNA sequences are complementary to the template)

• Non-template strand → Coding strand (mRNA sequences are identical to this)

How is newly synthesized RNA assembled?

Complementary base pairing with DNA template (AT/U[2]CG[3])

What catalyses transcription?

RNA polymerase catalyses the assembly of ribonucleotides and formation of phosphodiester bonds between the growing polynucleotide and incoming ribonucleotides → Forms the sugar-phosphate backbone

What is the direction of synthesis in transcription?

• 5’ to 3’ → 5’ phosphate group of incoming ribonucleoside triphosphate reacts with 3’OH group of the growing polynucleotide

• Template strand is read in the 3’ to 5’ direction

What happens during initiation of transcription?

1. Recognition and binding of general transcription factors (proteins) → RNA polymerase recruited at promoter

2. RNA polymerase unzips and separates the two strands of the double helix at the promoter by breaking H bonds between complementary base pairs → Template and non-template strand

How do free ribonucleotides from the nucleoplasm match up with the DNA template?

• Complementary base pairing (AU/T [2] CG [3])

• Synthesis in the 5’ to 3’ direction, moves along the template strand from 3’ to 5’

• As each nucleoside triphosphate is brought in, 2 terminal phosphates are removed

What happens at the end of transcription?

Termination: RNA polymerase has transcribed through specific DNA sequences known as termination sequences → mRNA chain is released → RNA polymerase dissociates

What is formed after transcription?

• pre-mRNA = RNA transcript formed immediately after transcription

• Produces functional mature mRNA

• Transcription unit of eukaryotic genes contains

  • Exons → Protein-coding sequences

  • Introns → Non-coding regions between exons

What happens during RNA processing in the nucleus?

1. Addition of the 5’methylguanosine cap

2. RNA splicing

3. Synthesis of 3’poly-A tail

How is the 5’methylguanosine cap added?

Methylated guanosine nucleotide is added to the 5’ end of the pre-mRNA molecule

What happens during RNA splicing?

• Intron sequences are excised and the exons are joined together → mature mRNA

  • Very precise points of excision determined by the sequences of nucleotides at the intron-exon boundaries

• Splicing is carried out by the spliceosome, an snRNA-protein complex (small nuclear RNA protein)

• Requires the hydrolysis of ATP

How is the 3’ end of the poly-A tail synthesized?

1. 3’ end of the pre-mRNA is cleaved enzymatically at a site about 10-35 nucleotides downstream of a highly conserved polyadenylation signal (AAUAAAA sequence)

2. Polyadenylation: Poly-A polymerase adds a series of adenosine monophosphate to form a poly-A tail

What is translation?

The process by which a sequence of ribonucleotides in an mRNA molecule is used as a template to synthesize a sequence of amino acids in a polypeptide chain

What occurs before translation?

• Amino acid activation catalyzed by aminoacyl-tRNA synthetases for each amino acid

• Each amino acid is covalently bonded to its specific tRNA with specific anticodon at its 3’CCA stem → aminoacyl tRNA

How does aminoacyl-tRNA synthetase work?

• Double specificity → Specific active sites complementary to conformation and charge of

  • Specific amino acid to be attached

  • Specific tRNA (Identity sites at acceptor stem and/or anticodon loop)

• With ATP

What happens during initiation of translation? (sites and subunits)

Binding of ribosome to mRNA (with tRNA) → Translation initiation complex → Translation begins at the start codon AUG

• Small subunit of ribosome and initiator tRNA carrying methionine assemble here

• Large subunit binds and completes a ribosome

• P site → Initiator aminoacyl-tRNA

• A site → Vacant for addition of the next aminoacyl-tRNA molecule

• Requires GTP

Where does polypeptide synthesis start and end during elongation?

• Starts at amino/N terminal

• Ribosome shifts one codon down the mRNA in the 5’ to 3’ direction

• Ends at carboxylic/C terminal

How does the next amino acid join the growing polypeptide chain during elongation of translation?

1. Next tRNA carrying the next amino acid binds to A site by complementary base pairing → Hydrogen bonds between anticodon and the codon on mRNA

2. Amino acid before dissociates from the tRNA it was originally bound to

3. Peptide bond formation between the two amino acids in A site ← Catalysed by peptidyl transferase

What occurs in each site of the large ribosomal subunit during elongation of translation as the ribosome moves one codon down?

• E site → 1st tRNA is released into cytosol (can be recycled)

• P site → 2nd tRNA, now carrying the growing polypeptide

• A site → Empty to receive 3rd tRNA with anticodon complementary to the third codon along mRNA

When does elongation stop?

Termination

1. Stop codon reaches A site → Specific proteins called release factors enter A site → Binding of release factors causes hydrolysis of the bond between polypeptide chain and the tRNA

2. Polypeptide is released from ribosome → Complete folding to assume to assume the necessary secondary and tertiary protein structures

3. Ribosome disassembles into subunits

What are the similarities between transcription and replication? (6)

• Occur within nucleus

• Complementary base pairing

• Unzipping and rewinding of DNA double helix

• Separation of parental strands occurs progressively in short segments

• Formation of phosphodiester bonds

• Involve proofreading mechanism for production of accurate nucleic acid chains

What are the differences between replication and transcription? (polymerase, NTP, template, base pairs, products)

• Replication → DNA polymerase, Transcription → RNA polymerase

• Replication → dNTP (DNA) and ribonucleotides NTP (RNA primer), Transcription → rNTP

• Replication → Both strands are templates, Transcription → 1 strand is a template

• Replication → AT GC, Transcription → AT/U CG

• Replication → 2 double stranded DNA molecules synthesized, Transcription → 1 single stranded RNA molecule synthesized

What are the differences between transcription and translation? (location, info transfer, enzymes, bonds, reading genetic message)

• Transcription → At nucleus of eukaryotes, Translation → At ribosomes of cytoplasm / Surface of rER

• Transcription → Info from DNA to mRNA, Translation → Info from mRNA to polypeptide

• Transcription → RNA polymerase, Translation → Peptidyl transferase and aminoacyl tRNA synthetase

• Transcription → Phosphodiester bond between ribonucleotides, Translation → Peptide bond between amino acids

• Transcription → Genetic message read 3’ to 5’, Translation → Read from 5’ to 3’