BIO 1A03 T2M1: Transcription

Theme 2

Genes

sections of DNA molecule that contain info that's transcribed into RNA copy

Central dogma

describes the flow of genetic information from DNA to RNA to protein, proposed by Francis Crick (late 1950s)

Gene expression

process of going from DNA to a functional product

Template strand

strand that RNA polymerase binds onto and makes complementary mRNA nucleotides

↳ AKA non-coding strand, minus (-) strand, antisense strand

Non-template strand

strand that RNA polymerase DOES NOT bind onto, but looks like the new mRNA transcript (same sequence but theres a T on the DNA strand, and U on the mRNA)

↳ AKA non-template strand, coding strand, plus (+) strand, sense strand

Transcription

turning DNA into RNA via mRNA

↳ happens in a transcription unit (containing ONE gene) → bc one gene = one protein

RNA polymerase

enzyme that helps with transcription

↳ attaches to promoter regions of DNA

Promoter region

region where the RNA polymerase and associated proteins bind to on DNA
↳ transcription is only initiated on only one strand, despite the promoter "region" being in both the strands (bc both strands are needed to recruit the proteins)
↳ found upstream @ 5' of GOI

Upstream

nucleotides towards the 5' end (3' to 5')

Downstream

nucleotides towards the 3' end (5' to 3')

5' end

has phosphate group

3' end

has hydroxyl group

Transcription steps in prokaryotes

composed of 3 main steps: initiation, elongation, termination

Holoenzyme

the resulting molecule composed of RNA polymerase + Sigma factor

Nucleotide

general term for nucleic acids components (nitrogenous base, pentose sugar, phosphate group)

Ribonucleotide

specific nucleotide where the pentose sugar is a ribose,

>> is the building block of RNA

TATA box

Promoter region in eukaryotic and archaea, characterized by the sequence 5'-TATA-3'
↳ includes the sequence: 5'-TATAAT-3'
-10 region (10 bases upstream)
↳ transcription takes place ~25 nucleotides downstream of TATA box
↳ this is an example of a consensus nucleotide: most common nucleotide sequences found in specific DNA/RNA locations
↳ another example: TTGCCA sequence → -35 region (35 bases upstream)

Consensus nucleotide

Most common nucleotide sequences found in specific DNA/RNA locations.

sigma factor

proteins that recognizes and facilitate the binding of RNA polymerase to promoter regions of DNA
↳ once transcription started, the sigma factor dissociates and the RNA polymerase continues transcription
⤷ prokaryotes have several different types of sigma proteins, each binding to a different promoters → determines what gene is transcribed at a time
↳ only thing necessary for prokaryotes to bind to promoter region
⇒ initiation of transcription occurs when RNA polycore enzyme binds to the sigma subunit to create holoenzyme

-10 region

the TATA box is -10 base pairs upstream (or towards the 5’ end of the template strand) of the start of transcription

-35 region

The region located 35 bases upstream of the TATA box, exemplified by the TTGCCA sequence.

actual process of transcription in prokaryotes (explained)

Initiation
- RNA poly attaches to promoter (starting point of DNA) - without the need for a primer
- RNA poly unwinds the DNA double helix → creating a transcription bubble ⇒ creating a template and non-template strand
↳ exposes:
Template strand (3′ → 5′) → used for RNA synthesis
Non-template strand (5′ → 3′) → not read, matches the RNA sequence (except U for T).
- Template strand is threaded through a channel inside the RNA polymerase, that leads to the active site → to begin building the RNA molecule transcript
↳ The RNA transcript is complementary to the template strand (opposite matched base pairs)

Elongation
- Ribonucleotide triphosphates (NTPs) enter another channel of RNA polymerase and moves towards the active site
- Complementary base-pairing occurs:
RNA polymerase reads the template strand 3′ → 5′
It brings in the complementary RNA nucleotides (A→U, T→A, C→G, G→C).
When the template contains adenine (A), the enzyme inserts uracil (U) instead of thymine.
Polymerization reaction occurs to form phosphodiester bonds - adding nucleotides to 3' end of the growing RNA molecule (RNA grows in 5' → 3')
- As RNA polymerase moves:
RNA polymerase keeps the template and non-template strand separate, so ribonucleotides (RNA building blocks) can pair with the template strand
-Passes template DNA through the active site channel, threads it out through another channel, and rewinds the DNA helix behind it

Transcription bubble

The structure created when RNA polymerase unwinds the DNA double helix during transcription.

Ribonucleotide triphosphates (NTPs)

The building blocks that enter RNA polymerase and are used to synthesize RNA.

Complementary base-pairing w RNA

The process where RNA polymerase reads the template strand 3′ → 5′ and brings in complementary RNA nucleotides (A→U, T→A, C→G, G→C).

Initiation of transcription in prok

Occurs when RNA polymerase enzyme binds to the sigma subunit and unwinds the double-stranded DNA helix

Polymerization reaction

process by which the enzyme RNA polymerase synthesizes an RNA strand by adding nucleotides one by one to a growing chain, forming phosphodiester bonds

--> Ribonucleotides are added to the 3' end of the new-elongating RNA transcript

1. Each incoming ribonucleoside triphosphate will correctly base pair with the template DNA's nitrogenous base

2. In order to link the sugar and phosphate of each new ribonucleotide, phosphodiester bonds must be made
   ↳ a phosphate group connects the 3'-carbon of one of the ribose sugar to the 5'-carbon of the next ribose sugar
   ↳ a lot of energy is required to create the phosphodiester bond → the energy comes from the incoming ribonucleoside triphosphate's phosphate bond energy
   - The 3' -OH group of the growing RNA attacks the high-energy phosphate bond of the incoming ribonucleotide and provides the energy to drive the polymerization reaction
   - The 2 phosphates of the incoming ribonucleotide is released and results in a phosphate-phosphate group (pyrophosphate)

what makes polymerization reaction of RNA irreversible

⇒ cleavage of the pyrophosphate (cutting) makes the polymerization reaction irreversible

Phosphodiester bonds

Bonds formed between the 3'-carbon of one ribose sugar and the 5'-carbon of the next ribose sugar, linking the sugar and phosphate of each new ribonucleotide.

Terminator sequence

DNA sequence that signals the end of a gene and triggers the termination of RNA synthesis

Rho-dependent termination

contains binding site for a "Rho factor" protein to bind onto the sequence and start "climbing" up the transcript towards the RNA polymerase
↳ once it reaches the polymerase at the transcription bubble, Rho pulls the RNA transcript and the DNA template strand apart ⇒ releasing the RNA transcript and ending transcription
↳ spot point: another sequence is found in Roh-dependent termination that causes RNA polymerase to pause... allowing Roh to catch up

Rho-independent terminator

↳ consists of inverted nucleotide repeat sequences on mRNA
↳ when the inverted repeat sequence is formed, the complementary nucleotides binds to each other (via Hydrogen bonds) to form a GC rich hairpin loop on the same mRNA strand
⤷ GC rich = guanine and cytosine complementary bases because they have 3 H-bonds between them, so stronger than AU bonds
↳ pauses the RNA polymerase and causes the release of the mRNA transcript

Inverted repeats

sequences of nucleotides that are ū followed downstream by a specific reverse complement (eg. AGCCCGC ……..GGCGGGCT) → that forms hairpin

3' -OH group

The hydroxyl group at the 3' end of the growing RNA that attacks the high-energy phosphate bond of the incoming ribonucleotide.

Pyrophosphate

A phosphate-phosphate group released during the addition of a ribonucleotide, which drives the polymerization reaction.

Why does coupling work in prokaryotes?

⇒ prokaryotic cells lack compartmentalization → ∴ can be temporarily and spatially coordinated
- lacks the presence of an nuclear envelop that separates the process of transcription and translation
- Prokarytoic genes are organized differently than eukaryotes → ∴ leads to different characteristics between mRNA transcript produced by prokaryotes at the end of the transcriptio

Active site channel

found within RNA polymerase enzymes where DNA template strand and nascent RNA thread thru —> allow nucleotide substrate to enter for RNA synthesis

Energy for polymerization

The energy required to create phosphodiester bonds, sourced from the phosphate bond energy of incoming ribonucleoside triphosphates.

Ribosome function in prokaryotes

The ribosome starts to make proteins while the other end (3') of the mRNA is still being transcribed.

Promoter in prok

DNA sequence that tells where the RNA polymerase should bind to start transcription.

General transcription factors

Proteins required for eukaryote's RNA polymerase to bind onto a promoter to start transcription.

RNA polymerase I

Transcribes genes for rRNAs (ribosomal RNA).

RNA polymerase II

Transcribes mRNA (messenger RNA) → serves as a template for protein production.

RNA polymerase III

transcribes genes for tRNAs (transfer RNA) + small regulatory RNA molecules
⇒ RNA polymerase I and III transcribe structural non-coding RNAs

Post-transcriptional modifications

modifications that must be done to the RNA transcript after transcription in order to enter into the nucleus
↳ includes: 5' cap + 3' polyAtail + splicing
↳ helps with stability of the mRNA, protection against ribonuclease enzymes that target the phosphodiester bonds, and help with the attachment of the ribosome and initiation of translation as it reaches the cytoplasm of the cell

5' cap modification

a modified guanosine attaches to the 5' end of the mRNA
↳ attaches via 5' to 5' triphosphate linkage
↳ the terminal 5' phosphate is removed from the mRNA molecule by a phosphatase enzyme and guanosyl transferase enzyme, and catalyzes the attachment of the of modified guanosine
7-methylguanosine: the modified guanosine used in 5' cap modification
↳ has a methyl group, attached to the 7th position of the guanine

↳ 7-methylguanosine

↳has a methyl group, attached to the 7th position of the guanine

7-methylguanosine

The modified guanosine used in 5' cap modification, with a methyl group attached to the 7th position of guanine.

PolyA tail

a bunch of adenine nucleotides added to the 3' end of the mRNA transcript
↳ follows the recognition of a polyadenylation signal sequence: AATAAA (that is transcribed from the DNA template strand near the end of the gene sequence)
↳ once signal is transcribed → mRNA is cleaved and polyA polymerase enzyme is able to add 150-200 adenine nucleotide bases to the 3' end of the RNA transcript

Polyadenylation: process of adding adenine nucleotides to the 3' end of the RNA transcript

Polyadenylation

process of adding adenine nucleotides to the 3' end of the RNA transcript

RNA Splicing

Process of removing introns and splicing together exons to make mRNA
↳ required to make sure the protein is in the correct sequence of amino acids
↳ occurs at specific short nucleotide sequences that are situated at the end of an intron

snRNPs

(small nuclear ribonucleoproteins) recognize and bind to regions where exons and introns meet

⤷ composed of snRNA and other proteins
⤷ 5 snRNPs interact with other proteins ⇒ forming spliceosome complex

Spliceosome complex

large RNA-protein complex that catalyzes removal of intros from nuclear pre-mRNA
⤷ able to recognize and form complementary base pairing with the nucleotides at the splice locations
⤷ catalyze reaction that allows a specific hydroxyl group on the nucleotide to attack and form new phosphodiester bond with nucleotide on the donor site
⤷ allows 5' end of intron attaches to the adenine in the intron to form lariat intermediate that forms loop
⤷ the 3' hydroxyl bond at the end of 5' exon, at donor, attacks the phosphodiester bond and make a new one between the 3' upstream of exon + 5' of downstream of exon
⇒ allows for the release of intro & splicing of exons

process of RNA splicing

- snRNPS assemble on the primary mRNA transcript ⇒ form spliceosome
- the intron forms a loop that breaks when a specific adenine nucleotide in the intro RNA attacks the 5' end of the intron
- The spliceosome mediates the breakage of the intron at the 5' end
- The free 5' end of intron attaches to the adenine in the intro ⇒ form lariat
- The free 3' end of exon 1 reacts with the 5' end of exon 2 ⇒ results in
- Breaking the 3' end of the intron
- Joining the 2 exons together with a covalent bond
- Lariat structure is degraded to nucleoside monophosphates

lariat

the loop created when introns join together to form spliceosome (composed of introns)

Alternative splicing

When only certain exons are used to form a mature RNA transcript

» allowing for multiple proteins from one gene.

Exons

Segments of mRNA that code to make proteins and will be spliced together.

Introns

Segments of mRNA that are non-coding and will be excised (removed).

Mature mRNA

The final mRNA result that gets to translation after all modifications.

Termination of RNA polymerase II

Termination of RNA polymerase II depends on polyA dependent mechanism of termination

↳ ∴ termination of transcription is coupled with mRNA maturation
↳ Modifications of the 3 prime mRNA transcript is coupled with the termination of transcription

termination of RNA polymerase I

Terminated using specific eukaryotic termination factor - similar to "prokaryotic dependent termination"

termination of RNA polymerase III

Terminated after transcription sequence - similar to "roh-independent termination" in prokaryotes

Nuclear pore

⤷ can transport RNA, proteins, carbohydrates, important signalling molecules,

protein line channels that pass through both membranes the nuclear envelope, allowing molecules to move in and out of the nucleus

what happens to the lariat

Broken down into individual nucleotides to be recycled after excision.

W. Gilbert

Proposed the idea of introns and exons.

Purpose of introns

Allow for alternative splicing, contributing to genetic diversity and enhancing crossing over during meiosis.