Chapter 8: Transcription and RNA Processing

Learning Objectives

Compare and contrast the structure of DNA and RNA; describe the classes of RNA molecules involved in gene expression.
Diagram transcription, including the enzymes involved in transcription initiation, elongation, and termination.
Describe the location and structure of consensus sequences in eukaryotic promoters; distinguish between promoters, enhancers, and silencers.
Determine the position of the promoter using a band shift assay.
Given a DNA template, indicate the direction of transcription and predict the mRNA sequence including polarity.
Given an mRNA sequence, predict the DNA template and coding sequence and indicate the location of the promoter sequence.
Detail the three steps in eukaryotic post-transcriptional processing: 5’ capping, 3’ polyadenylation, and intron splicing (including alternative splicing).

The Central Dogma: Transmission of Information

Gene: DNA → mRNA → Protein
DNA Template Strand Example: 3'-TACCACAACTCG-5'
Transcription: DNA is transcribed into mRNA.
mRNA Example: 5'-AUGGUGUUGAGC-3'
Triplet code words are present in mRNA.
Translation on ribosomes: mRNA is translated into a sequence of amino acids.
Amino acid sequence example: met-val-leu-ser.

Basics of Transcription

DNA is double-stranded and serves as a template.
RNA: Ribonucleic acid; the product of transcription.
Transcription: Process of copying information from DNA to RNA.
RNA Transcript: Single-stranded.

RNA Structure

RNA uses ribose sugar instead of deoxyribose.
Ribose has an OH group on the 2' carbon, while deoxyribose has only H.
Deoxyribose chemical structure:

\begin{aligned}
& \text{5' HOCH2} \
& \text{4' C-H} \
& \text{3' C-H} \
& \text{2' C-H} \
& \text{OH H}
\end{aligned}
Ribose chemical structure:

\begin{aligned}
& \text{5' HOCH2} \
& \text{4' C-H} \
& \text{3' C-H} \
& \text{2' C-OH} \
& \text{OH OH}
\end{aligned}

RNA Nucleobases

RNA uses uracil (U) instead of thymine (T).
Purine nucleotides: Adenosine 5'-monophosphate (AMP), Guanosine 5'-monophosphate (GMP).
- Chemical structure of Adenosine 5'-monophosphate (AMP):
  
  \text{Phosphate - Nucleotide base}
  
  \text{H₂C 5'}
- Chemical structure of Guanosine 5'-monophosphate (GMP):
  
  \text{Phosphate - Nucleotide base}
  
  \text{H₂C 5'}
Pyrimidine nucleotides: Uridine 5'-monophosphate (UMP), Cytidine 5'-monophosphate (CMP).
- Chemical structure of Uridine 5'-monophosphate (UMP):
  
  \text{Phosphate - Nucleotide base}
  
  \text{H₂C 5'}
- Chemical structure of Cytidine 5'-monophosphate (CMP):
  
  \text{Phosphate - Nucleotide base}
  
  \text{H₂C 5'}

Thymine vs. Uracil

Thymine:
$\text{Chemical Formula: } C5H6N2O2$
Uracil:
$\text{Chemical Formula: } C4H4N2O2$
Uracil still forms two hydrogen bonds with adenine.

Informational RNAs

Contain information encoded in DNA to make proteins.
Includes mRNA (messenger RNA).
Process:
- Transcription start site is downstream of the promoter.
- Addition of cap to the 5' end.
- 3' cleavage.
- Addition of poly(A) tail.
- Splicing to remove introns.
Mature mRNA consists of exons with a poly(A) tail.
Untranslated regions (UTRs) are present at the 5' and 3' ends.

Functional RNAs

Have an active function beyond carrying information for translation.
Examples:
- transfer RNA (tRNA): bring amino acids to ribosomes during translation.
- ribosomal RNA (rRNA): structural components of ribosomes.

Types of RNA

mRNA (messenger): Intermediate molecules for transferring information from DNA to protein.
rRNA (ribosomal): Functional RNA molecules that are components of the ribosome.
tRNA (transfer): Functional RNA molecules that serve as adapters in translation.
snRNA (small nuclear): Functional RNA molecules involved in the removal of introns from pre-mRNAs.
snoRNAs (small nucleolar): Required for rRNA processing.
Various other functional RNAs: microRNAs, small-interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), long non-coding RNAs (lncRNAs).

Transcription

RNA polymerase opens the DNA double helix and uses one strand as a template for transcription.
The strand used as the template is characteristic for that gene.
RNA polymerase elongates the RNA strand using base complementarity as a guide.
RNA polymerase elongates the RNA in the 5' → 3' direction.
DNA is the template for transcription.

Conventions of Transcription

Gene: The physical unit of heredity, composed of a DNA sequence that is transcribed and encodes for a protein or another functional transcript.
Types of genes:
- Protein-coding genes: transcribed to produce mRNA encoding a protein.
- RNA genes: transcribed to produce a functional RNA.

Protein Coding Genes

Protein-coding genes are transcribed to produce a mRNA that will encode a protein.
Key elements:
- Promoter region: where RNA polymerase binds.
- Coding region: the portion of the gene that is transcribed into mRNA and translated into protein.
- Termination sequence: signals the end of transcription.
Upstream and downstream regions relative to the transcription start site (+1).
Coding (nontemplate) strand and template strand.
Transcription direction: indicated.

Example of Transcription

Coding strand: 5'-AGCTGGACATTGGCCATG-3'
Template strand: 3'-TCGACCTGTAACCGGTAC-5'
RNA transcript: 5'-AGCUGGACAUUGGCCAUG-3' (same as the coding strand but with U instead of T).

RNA Polymerases in Eukaryotes

They resemble polymerases in prokaryotes.
RNA Polymerase I: transcribes rRNA.
RNA Polymerase II: transcribes mRNA.
RNA Polymerase III: transcribes tRNA.
Table of RNA Polymerase Protein Subunits:
- Bacterial Core: β, β’.
  * Archael Core: A’/A”, B, D, L, K [+6 other subunits].
- Eukaryotic Cores: * RNA pol I: RPA1, RPA2, RPC1, RPC2, RPC5, RPC9, RPB6 [+9 other subunits].
  - RNA pol II: RPB1, RPB2, RPB3, RPB11, RPB6 [+7 other subunits].
  - RNA pol III: RPC1, RPC2, RPC5, RPC9, RPB6 [+11 other subunits].

Eukaryotic Promoters

Eukaryotic promoters are diverse, but there are still consensus sequences.
Some sequences are more conserved than others.
Different genes have different consensus sequences in their promoters.
These consensus sequences are important for binding transcription factors.

Consensus Sequence Example

From 10 genes in E. coli, deduce the consensus sequence for the -35 region and the -10 region.
-35 region consensus sequence: TTGACA
-10 region consensus sequence: TATAAT

Transcription Factors

TFIID: Transcription factors that attract RNA Pol II.
TFIID = TATA binding protein (TBP) and TAF (TBP-associated factor).
The complete initiation complex guides RNA Pol II to +1, where it will initiate transcription.

Regulatory Sequences

Enhancers: Can be tens of thousands of base pairs away, upstream or downstream, and interact with transcription factors via a protein bridge.
Silencers: Behave similarly to enhancers but act to repress transcription.
Other kinds of regulatory sequences are required for transcription in eukaryotes.

Regulation of Transcription Factors

Some genes require specific transcription factors, which themselves have tightly regulated synthesis.
Sometimes transcription factors need to be activated by way of a signal transduction pathway.
An external stimulus (growth factor, hormone, light) which releases a transcription factor that can then bind to an enhancer or promoter.

Cis-Regulatory Elements and Trans-Acting Factors

Cis-regulatory elements: Regions of DNA (enhancer, promoter, coding region) that regulate the transcription of a gene.
Trans-acting factors: Transcription factors, activators, repressors.

Band Shift Assay

How can we identify the DNA sequences that bind to proteins?
Control: DNA with no protein added.
Experimental: DNA with transcription protein added.
If promoter consensus sequences are in the DNA fragment, the proteins will bind to them.
Slower migration indicates a higher molecular weight produced by binding of transcriptional proteins to promoter sequences on DNA.

Band Shift Assay Example

Transcription factor bound.
Transcription factor not bound.
Changed promoter sequence differently in each, added transcription factor proteins, and performed gel electrophoresis.
Promoter sequence: 5'-AGTCGT-3' → Binding occurs.

Problem Example: Promoter Sequence Importance

Original sequence: 5'-AGTCGT-3'
A: 5'-AGCCGT-3' → Change in 3rd nucleotide (T to C) caused the factor to not bind. T is important.
B: 5'-AGTGGT-3' → Change in 4th nucleotide (C to G) did not change binding. C or G is not as important.

More Promoter Sequence Examples

C: 5'-AGTCAT-3' → Change in 5th nucleotide (G to A) caused the factor to not bind. G is important.
D: 5'-ACTCGT-3' → Change in 2nd nucleotide (G to C) did not change binding. G or C is not as important.

Example Problem: Determining Important Nucleotides

You synthesize the promoter sequence, which has the consensus sequence 5'-AGTCGT-3', but make four different variants (A through D, below), each one with a different DNA sequence. You then add the transcription factor to this DNA and run the DNA-protein mixture on a gel to see how they move together. Below is the result of your band shift assay. Which position(s) in the DNA sequence are crucial for binding of the transcription factor?
5'-AGTCGT-3'
Transcriptions:
- A: 5'-AGCCGT-3'
- B: 5'-AGTGGT-3'
- C: 5'-AGTCAT-3'
- D: 5'-ACTCGT-3'

Transcription Termination and Processing

RNA pol I: polyU region destabilizes transcription.
RNA pol III: transcription terminating factor I (TTFI) binds to a consensus sequence that stops transcription.
RNA Pol II: termination of mRNA transcription is not well understood, but these pre-mRNA transcripts are heavily processed!
Addition of the 5' cap: methyl transferase adds a methyl group to the 7-nitrogen to form 7-methylguanosine; guanylyl transferase performs these three steps.

5' Capping

Methyl transferase adds a methyl group to the 7-nitrogen to form 7-methylguanosine. Guanylyl transferase performs these three steps.
5' Cap is important for:
- prevention of degradation of the mRNA.
- transport across the nuclear envelope.
- facilitating intron splicing.
- orienting the mRNA for translation.

3' Polyadenylation

Adding the 3' polyA to the pre-mRNA.
PAP = polyadenylate polymerase.
polyA binding proteins (PAB) facilitate polyA lengthening.
20-200 adenines are added.
cleavage factors CPSF = cleavage and polyadenylation specificity factor.

Importance of Poly(A) Tail

polyA tail is important for:
1. prevention of degradation of the mRNA.
2. transport across the nuclear envelope.
3. orienting the mRNA for translation.

Splicing

Splicing: the removal of intron sequences.
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The precision of splicing is very important!

Splicing - Signal Sequences

5' splice site, branch site, 3' splice site.
Exon 1 - Intron 1 - Exon 2
Consensus sequences:
- 5' splice site: 5' AGGU AGU 3'
- Branch site: PyNPyPyPuAPy (G)PyNC (Branch point adenine).
snRNP U1 binds 5' splice site, and U2 binds branch site.
snRNP = "snurp" = small nuclear ribonucleoprotein

Splicing - Formation of the "Lariat"

snRNPs U4, U5, and U6 bind to complex and form the inactive spliceosome.
A lariat intron structure forms.

Lariat Intron Formation

Lariat intron forms by a 2'-5' phosphodiester bond between the 5' guanine and the branch point adenine.

Splicing - Transesterification Reactions

Two transesterification reactions are needed to excise an intron.
- 2' OH of the branch site bonds with P at the 5' splice site.
- New OH on the 5' G bonds to P at the 3' splice site.
Connect exons and excise intron.

Alternative Splicing

Alternative splicing allows multiple mRNA molecules (and therefore proteins) to be produced from a single gene.
Alternative splicing is controlled by splicing regulatory proteins that bind to exons.

Coupling Transcription with mRNA Processing

(PIC) carboxyl terminal domain of RNA Pol II acts as a mediator for pre-mRNA processing.

5' Capping, Elongation, Splicing, and Polyadenylation

RNA pol II initiates transcription. PIC dissociates, leaving the pre-mRNA processing proteins on the CTD. CAP proteins carry out 5' capping.
Capping proteins dissociate and pre-mRNA elongates.
Spliceosome complexes affiliate with pre-mRNA with the aid of SF proteins. Intron splicing takes place as RNA pol continues elongation of mRNA.

Transcription Termination and Polyadenylation

Polyadenylation proteins identify the pA signal sequence and carry out polyadenylation. Transcription terminates. Splicing continues to completion.

Self-Splicing introns

Two transesterification reactions occur, but the mRNA itself catalyzes the reactions without the need for a spliceosome.
Several kinds of self-splicing introns exist and they occur in mRNA, tRNA, and rRNA.