BIOL 3000 Transcription and Intron Processing

Central Dogma

Gene (genotype) → mRNA (intermediate) → “Protein”/polynucleotide (phenotype)

Transcription

DNA to mRNA

Where: cell nucleus (eukaryotes)

When: Either G1/S or G2/M (waves of transcription that coincides with different transition points during the cell cycle)

Doesn’t happen in mitosis

Can sometimes happen in S phase

“Waves” of transcription

Times that make lots or little mRNA

Basic Rules of Transcription

Only one of the two DNA strands serves as a template: template and coding strands

Selective process

Always in 5’→3’ direction; nucleotides added at the 3’ growing tip

Results in an RNA compliment (anti-parallel and unidirectional)

Template strand

The DNA strand that mRNA is built from

The strand that the enzymes are actually reading

Coding strand

“Exact” same sequence of nucleotides in the mRNA except T and ribose. Making a complimentary strand T-U

Selective process of transcription

Each gene has its own transcription protocol (has its own promoter)

Steps of Transcription

0. Pre-recognition: accessibility to DNA

1. Recognition: pre-initiation complex formation

2. Initiation: binding of RNA polymerase complex

3. Elongation: movement of RNA Pol II and formation of mRNA

4. Termination: cleavage of new transcript

Pre-recognition

DNA access

DNA is packaged in the nucleus as heterochromatin which we cannot make protein from this because we cannot get to the DNA

So it must be converted to euchromatin before transcription

Part of Gene Expression Regulation

Nucleosome (10nm fiber)

Nucleosome (10nm fiber)

Histone core is positively charged, and the DNA is negatively charged

Lys (K) acetylation

Histone acetyl transferase (HAT)

Histone deacetylase (HDAC)

Allows the tail to open and close, releasing and loosing the DNA

Histone acetyl transferase (HAT)

Transfer acetyl group to the histone

This tightens the tail and holds onto the DNA

Increases the positive charge of the nucleosome

Histone deacetylase (HDAC)

Removes the acetyl group from the histone

This looses the tail and loosens the DNA and makes it able to move

Decreases the positive charge of the nucleosome

Recognition: pre-initiation complex (PIC) formation

Forms a large complex of ~100 proteins (PIC) required for RNA Polymerase II to bind to

TATA Binding Protein (TBP) binds the promotor region (TATA Box), once bound, it recruits the rest of the pre-initiation complex. Then the mediator complex arrives

General Transcription Factors (TF IID) recruited by TBP

Initiation: binding of RNA polymerase complex

Other transcription factors (TF IIB, TF IIF, TF IIH) and RNA Pol II recruited to complex

Mediator complex: ~20 proteins

RNA Pol II binds to the Template Strand via its Active Site and is NOT on

Mediator complex

ATPase and helicase activity unwind DNA so that polymerase can read

One turn of DNA unwinds and forms a transcription bubble

RNA Pol II

Unphosphorylated at its carboxyl end (CTD)

Elongation: Movement of RNA Pol II and formation of mRNA

RNA Pol II is phosphorylated at its carboxyl end (CTD) by Mediator Complex

RNA Pol II traverses the template strand and creates an RNA copy

RNA Pol II traverse the template strand from 3’→5’

Exact copy of the coding strand (except that Thymines are replaced with Uracils, and the nucleotides are composed of ribose (5-carbon) sugar

READS

3’→5’

WRITES

5’→3’

Termination: cleavage of new transcript

Two new protein complexes carried by CTD recognize the poly-A signal (AAUAAA)

Once poly-A signal is made, the polymerase can stop

CPSF (cleavage and polyadenylation specificity factor)

CSTF (cleavage stimulation factor)

Other proteins recruited to carry out cleavage

Post transcriptional processing can now occur

Image of Transcription

Basic Summary of Transcription

• DNA to mRNA (specifically hnRNA)

• Makes RNA 5’ - 3’ (ALWAYS ADDING TO THE 3’ END)

• RNA polymerase

  • Immature mRNA consists of BOTH exons and introns

The two types of post-transcriptional regulation

5’ capping

3’ polyadenylation

5’ Capping

A guanine group is added to the 5’ end of the growing RNA chain at about 30 nucleotides long. This group caps the enzymes copying the DNA.

Happens co-transcriptionally

Functions of 5’ guanine cap

• Regulates nuclear export

• Promotes translation (helps the ribosome recognize the message)

• Prevents the degradation of mRNA in the cytoplasm

• Involves in intron splicing

3’ Polyadenylation

The RNA is cleaved by ribonuclease downstream of the AAUAAA site. A poly(A) polymerase adds adenine ribonucleotides to the 3’ end of the RNA molecule. The enzyme is not dependent on the template and is up to 200 bases.

Functions of 3’ Polyadenylation

• Enhances the stability of the RNA molecule

• Regulates transport to the cytoplasm

RNA processing

Co-transcriptionally AND post-transcriptionally

Splicing

The mechanism by which introns are removed

Introns

Intervening sequences of RNA not expressed in proteins

Exons

Retained in mature mRNA and are the expressing sequences

Splicesome

Protein/RNA complex that directs and ensures proper RNA splicing. Responsible for removal of introns from transcribed mRNA

Responsible for both cleavage of the intron from the RNA and ligation of the remaining exons

hnRNA

Pre-mRNA

Immature single stranded mRNA present in the nucleus (contains introns)

snRNA

“Snurps”

Small nuclear RNA molecules associated with specific ribonuclear proteins playing essential role in splicing

[U1,U2, U4, U5, U6]

mRNA

“Mature mRNA”

RNA that is translated to become a polypeptide chain

Four Distinct Types of Introns

1. Introns in protein coding genes, removed by splicesomes

2. Introns in tRNA genes, which are removed by proteins

3. (Group 1) Self-splicing introns, which catalyze their own removal from mRNA, tRNA, and rRNA precursors using guanosine-5’-triphosphate (GTP), or another nucleotide cofactor (ribozymes)

4. (Group 2) Self-splicing intros, which do not require GTP in order to remove themselves but do require assistance from proteins

Intron

Intervening sequences

Has GU, AG, polypyrimidine tract, and branch point sequence

GU nucleotide sequence

At the 5’ splice site (donor site)

AG nucleotide sequence

At the 3’ splice site (acceptor site)

Polypyrimidine tract (PPT)

Just upstream of the 3’ splice site promotes the assembly of the spliceosome

It comes before the AG

Branch point sequence (UACUAAC)

The binding site for the snRNP-U2

Spliceosome Assembly (steps 1-5)

1. U1 binds to 5’ splice site

2. U2 binds to BPS

3. Trimer of U4, U5, and U6 recruited to 5’ splice site

4. U1 and U4 dissociate from hnRNA leaving U5 and U6 bound

5. U2 and U6 associate

Spliceosome Assembly (steps 6-9)

6. “Lariat” of hnRNA is formed from the intron

7. 5’ splice site is cleaved and U5 binds to 3’ splice site

8. 3’ splice site is cleaved and U5 ligates exons together via ATP hydrolysis

9. snRNP’s are released along with spliced intron

Alternative Splicing

A regulated process that results in a single gene coding for multiple proteins

It picks and chooses introns

It increases genetic diversity

The mechanism of removal is the same as the spliceosome

Under the control of specific factors

Trans-Acting Factors

Usually proteins that control gene expression and affects the DNA

Cis-Acting Factors

DNA sequences in the vicinity of a gene required for gene expression. Doing something to a nucleic acid

Primary splicing defects

Sequences in the pre-mRNA important for splicing are mutates (cis-activating factors)

Secondary splicing defects

Regulatory factors essential for splicing are mutated (trans-activating factors)

If the X protein acts on the RNA and covers the AG, then there will be more protein Z than protein Y