From DNA to Protein: Transcription and Translation

From DNA to Protein

• The lecture covers the flow of genetic information from DNA to protein, focusing on transcription and translation.

Central Dogma

• DNA serves as the blueprint for RNA synthesis, also known as transcription.
• RNA acts as an order form for protein synthesis, also as translation.
• Proteins are the functional parts made from amino acids

Regulation of Gene Expression

• Gene expression involves two main steps: transcription (DNA to RNA) and translation (RNA to protein).

Eukaryotes vs. Prokaryotes

• In eukaryotes, transcription occurs in the nucleus where pre-mRNA is synthesized and then processed into mRNA.
• Post-transcriptional processing is needed for mRNA production
• Eukaryotes have mitochondria and chloroplasts
• In prokaryotes, transcription happens in the cytoplasm.

Gene

• A gene is a fundamental hereditary unit carrying information from one generation to the next.
• Genes are sections of DNA within a chromosome coding for protein synthesis.
• A basic gene structure consists of a promoter, RNA coding sequence, and terminator.

Gene Structure Differences

• Prokaryotic genes have a coding region.
• Eukaryotic genes contain coding regions (exons) and noncoding regions (introns).

Gene Size and Structure

• The human β-globin gene consists of approximately 2000 nucleotide pairs.
• The human Factor VIII gene consists of approximately 200,000 nucleotide pairs.

Bacterial RNA Polymerase

• Signals in the nucleotide sequence indicate where bacterial RNA polymerase should start and stop transcription.

Transcription

• Transcription is the process where genetic information from DNA transfers into RNA through RNA synthesis.
• Transcription produces RNA and is complimentary to one strand of DNA.

Transcription in Prokaryotes

• Takes place in the cytoplasm
• DNA is unwound and RNA is synthesized by RNA polymerase
• The RNA coding sequence transcribes into mRNA

Transcription in Eukaryotes

• Transcription occurs in the nucleus.
• The RNA is synthesized in two steps
• Pre-mRNA is produced through transcription of eukaryotes. Then pre-mRNA undergoes RNA processing to produce messenger mRNA (mRNA).

Process of Transcription

• Transcription proceeds through three steps: initiation, elongation, and termination.

Initiation of Transcription

• Initiation begins at the promoter, a DNA sequence that includes CAAT, TATA, and GC boxes.
• Promoters are located at the 5' end of a gene.

RNA Polymerases

• RNA polymerase I synthesizes pre-rRNA (45S).
• RNA polymerase II synthesizes pre-mRNA, snRNA, and microRNA.
• RNA polymerase III synthesizes tRNA, rRNA (5S), and other microRNA.
• RNA polymerases IV & V synthesize plant's RNA.
• Other polymerases synthesize mitochondrial RNA, chloroplast RNA, and prokaryotic RNA.

DNA Template

• Only one DNA strand acts as a template for RNA synthesis.
• The promoter determines which DNA strand transcribes.

Template Determination

• When the promoter is on the left, the lower strand is the template, and the upper strand is the coding strand.
• When the promoter is on the right, the upper strand is the template, and the lower strand is the coding strand.

Gene Direction

• Genes can be located on both DNA strands.
• RNA transcript for gene b reads from right to left
• RNA transcript for gene a reads from left to right

Elongation of Transcription

• After RNA polymerase starts RNA synthesis, most initial transcription factors are released.
• Elongation factors help RNA polymerase unwind DNA further and stabilize the new pre-mRNA.

Termination of Transcription

• Termination occurs when RNA polymerase encounters a specific nucleotide sequence termination signal.
• In eukaryotes, the most common termination sequence is AATAAA.

Transcription Summary

• RNA polymerase binds to the promoter and unwinds local DNA area
• RNA synthesis begins with initiation
• RNA elongation follows in the 5' to 3' direction.
• Transcription ends at the termination signal.

Replication and Transcription

• Both use one strand of DNA as a template and follow the principle of complementarity.
• Transcription produces RNA, which doesn't remain attached to DNA and are copied from a limited region.
• Transcription doesn't need a primer.
• Amount of RNA is controlled per gene expression.
• Eukaryotic RNAs undergo major changes before leaving the nucleus.

Post-Transcriptional Modification

• post-transcriptional mRNA processing readies mRNA for translation

Pre-mRNA Processing

• Pre-mRNA processing occurs only in eukaryotic cells
• Primary transcript RNA (pre-mRNA) converts to mature messenger RNA (mRNA) in this process.
• Processing takes place in the nucleus during transcription.

5' Modification of pre-mRNA

• RNA capping and polyadenylation occur during pre-mRNA processing.
• Capping involves adding a "cap" and occurs at transcription's beginning.
• Capping takes place after the growing RNA strand reaches ~25 nucleotides.

Mechanism of Capping

• Capping enzymes attach a 7-methyl-guanosine "cap" to the transcript's 5' terminal base through a 5' to 5' triphosphate bridge.

Significance of the 5' Cap

• Regulates nuclear exporting.
• Prevents degradation by exonucleases.
• Promotes the initiation of translation.
• Promotes 5' proximal intron excision in splicing.

3' Modification of pre-mRNA

• Polyadenylation occurs through the addition of a poly-A tail.
• This step occurs at the end of transcription upon reaching the termination signal.

Mechanism of Polyadenylation

• An endonuclease recognizes the termination signal and cleaves the RNA.
• Poly(A) polymerase adds ~200 adenines at the 3' end of the mRNA.

Significance of the 3' Tail

• The poly-A tail differentiates mRNA from other types of RNA, it also ensures proper intron splicing.
• mRNA without a poly-A tail cannot leave the nucleus

Pre-mRNA Splicing

• Pre-mRNA splicing removes introns from pre-mRNA and connects exons to form a continuous coding sequence.
• Splicing occurs in the nucleus
• Involves spliceosomes, snRNPs and proteins

Special Nucleotide Sequences

• Specific nucleotide sequences signal the start and end of an intron.

Pre-mRNA Splicing Steps

• Occurs after the complete synthesis and cap of pre-mRNA.
• Catalyzed by a spliceosome protein complex.
• Includes proteins (endonuclease and ligase) and small nuclear ribonucleoproteins (snRNPs).

Mechanism of Pre-mRNA Splicing

• The RNA portion of snRNPs base-pairs with sequences to signal splicing.
• Additional snRNPs bind, assembling the spliceosome resulting in spliced products.

A Lariat Structure

• Branch point adenine attacks the 5' splice site.
• The intron’s cut end loops to the adenine and forms a loop.
• Forming a loop in the intron

Significance of Splicing

• Splicing removes non-coding introns from RNA and produces a mature mRNA with only protein-coding exons.

Alternative Splicing

• Alternative splicing allows different combinations of exons creating multiple forms of protein.

Exon and Intron Length

• Exon and intron length have a range and vary between different species.

Post-transcriptional Summary

• The post-transcriptional modifications of pre-mRNA lead to a change within the nucleus

mRNA Differences

• Prokaryotic mRNA doesn't have a 5' cap or poly-A tail
• Eukaryotic mRNA has a 5' cap and a poly-A tail

mRNA and Nuclear Pore

• "Export-ready" mRNA molecules transport throughout a nuclear pore in the membrane.

Translation

• Translation is the process where information from mRNA transfers to amino acids to create a polypeptide.
• Protein synthesis through is a result of polypeptide structure and synthesis

Translation location

• In eukaryotes, translation happens outside the nucleus in the cytoplasm
• In prokaryotes, transcription happens in also happens in the cytosol

Molecular Components

• Essential components for translation include: mRNA, amino acids, tRNAs, ribosomes, enzymes, and energy.

Codon

• Codons are part of a mRNA molecule to code for protein synthesis.
• Each codon has three nucleotides and each codon makes on type of amino acid.

Genetic Code Properties

• The genetic code is universal where translation is a set of rules to express it.
• Non-overlapping groups are required.
• There are no spaces during coding.
• Has redundancy.
• 64 possible codons but only 20 amino acids
• Has a start codon.
• Has three non-coding stop codons.
• Not all amino acids have the same amount of codons (some are more common)

Reading Frame

• The start codon typically initiates translation after the mRNA binds to the Ribosomes.

Translation Reading Frames

• There are three possible outcomes that translate nucleotide sequence to amino acid sequence with varying amino acid outcomes.

tRNA Binding site and Structure

• Three nucleotides on the cloverleaf structure pair with the mRNA
• Amino acids attach at the 3' end

Aminoacyl-tRNA Synthetase

• The enzyme causes a high energy bond for amino acids to bind
• tRNA synthetase links the amino acid and mRNA codon in the 5' end

Decoding by Ribosomes

• The mRNA message decodes by Ribosomes to translate proteins

Eukaryotic Ribosome

• Made of proteins and rRNA, eukaryotic ribosome is composed of small and large subunits for translation.

RNA binding sites

• The ribosome contains an E site, P site, and A site.
• mRNA binding also takes place, near the complex

Steps of Translation

• Aminoacyl-tRNA binds to the A site.
• Peptide and peptide bonds come in forming the ribosome
• Translocation of unit causes shift in RNA molecule

Specific Codons

• All three codons in mRNA Signal the Ribosome to Start and to Stop Protein Synthesis
• Translation happens when the AUG codon is reached.
• An initiator tRNA binds (methionine (Met.)

Translation Steps

• The elongation and transfer of tRNA to build the protein synthesis. Proteins then undergo a post shift in position from this translation.

Inhabitors

• Some antibiotics are inhabitors to expression of proteins