Difficulty: Intermediate | Prerequisites: Part 1 of these notes (gene expression basics, the genetic code)
This section zooms into the first half of gene expression: how a gene's DNA sequence is transcribed into RNA, and how that RNA is then processed before it leaves the nucleus. Eukaryotic mRNA undergoes extensive modification (capping, tailing, splicing) that bacterial mRNA does not. Understanding these modifications explains why eukaryotic gene expression is more complex and more flexible than bacterial gene expression.
RNA polymerase reads the template strand of DNA (3' to 5') and builds an mRNA strand (5' to 3'). In eukaryotes, the pre-mRNA receives a 5' cap and a poly-A tail, and introns are spliced out by the spliceosome. These processing steps protect the mRNA, help it leave the nucleus, and allow one gene to produce multiple proteins through alternative splicing.
RNA polymerase
The enzyme that pries open the DNA double helix and assembles RNA nucleotides complementary to the template strand, building in the 5' to 3' direction. Unlike DNA polymerase, it does not need a primer.
Promoter
A specific DNA sequence where RNA polymerase attaches and begins transcription. Think of it as the "start here" sign on the DNA.
Terminator
A DNA sequence that signals the end of transcription. In bacteria, the transcribed RNA sequence itself triggers the polymerase to detach.
Transcription unit
The stretch of DNA from the promoter to the terminator that is transcribed into a single RNA molecule.
Transcription factors
Proteins (in eukaryotes) that must bind to the promoter before RNA polymerase II can attach. They mediate and regulate the start of transcription.
Transcription initiation complex
The complete assembly of transcription factors and RNA polymerase II on the promoter. Once formed, the polymerase unwinds the DNA and begins transcribing.
TATA box
A crucial promoter DNA sequence (roughly TATAAA) located about 25 nucleotides upstream of the transcription start point in many eukaryotic genes. It helps position RNA polymerase correctly.
Polyadenylation signal sequence
A DNA sequence that, once transcribed, produces the RNA signal AAUAAA. This signal triggers cleavage of the pre-mRNA and the addition of the poly-A tail.
5' cap
A modified guanine nucleotide added to the 5' end of the pre-mRNA shortly after transcription begins. It protects the mRNA, helps it leave the nucleus, and helps ribosomes attach.
Poly-A tail
A stretch of 50 to 250 adenine nucleotides added to the 3' end of the pre-mRNA after cleavage. It shares the same protective and export functions as the 5' cap.
Untranslated regions (UTRs)
Sequences at the 5' and 3' ends of the mRNA that are not translated into protein but have functions such as ribosome binding. They sit between the cap/tail and the start/stop codons.
Pre-mRNA (primary transcript)
The initial RNA transcript in eukaryotes, before processing. It contains both introns and exons, plus the sequences that will become the UTRs.
RNA splicing
The process of removing introns from pre-mRNA and joining the remaining exons into a continuous coding sequence.
Intron (intervening sequence)
A non-coding segment of nucleic acid that lies between coding regions. Introns are transcribed into pre-mRNA but removed before translation.
Exon
A coding segment of nucleic acid that is retained in the final mRNA and is usually translated into amino acids. The UTRs at each end are also considered exons even though they are not translated.
Spliceosome
A large complex of proteins and small RNAs that carries out RNA splicing. Its RNA components help recognise splice sites and catalyse the splicing reaction.
Ribozyme
An RNA molecule that functions as an enzyme. The discovery of ribozymes showed that not all biological catalysts are proteins. In some organisms, intron RNA can catalyse its own excision.
Alternative RNA splicing
The inclusion of different combinations of exons during splicing, allowing a single gene to produce multiple different mRNAs and therefore multiple different polypeptides.
Domains (protein)
Discrete structural and functional regions of a protein. Different exons often code for different domains, and exon shuffling during evolution can recombine domains into novel proteins.
Exon shuffling
An evolutionary process in which introns facilitate recombination between exons of different genes, potentially creating proteins with new combinations of functional domains.
RNA polymerase pries open DNA, reads the template strand 3' to 5', and builds RNA 5' to 3'
RNA polymerase does not need a primer (unlike DNA polymerase)
Bacteria have one type of RNA polymerase (makes mRNA and ribosomal RNA)
Eukaryotes have three RNA polymerases in the nucleus:
RNA polymerase I: rRNA
RNA polymerase II: pre-mRNA (the one you need to know best)
RNA polymerase III: tRNA and other small RNAs
Initiation
The promoter includes the transcription start point and extends several dozen nucleotide pairs upstream
In bacteria, part of the RNA polymerase itself recognises and binds the promoter directly
In eukaryotes, transcription factors must bind the promoter first; only then does RNA polymerase II attach
The TATA box (about 25 nucleotides upstream of the start point) is a key promoter element in eukaryotes
The full assembly of transcription factors + RNA polymerase II on the promoter = the transcription initiation complex
Once the complex is assembled, the polymerase unwinds the DNA and begins synthesising RNA at the start point
Elongation
RNA polymerase moves along the DNA, untwisting about 10 to 20 nucleotides at a time
New RNA nucleotides are added to the 3' end of the growing strand
Behind the polymerase, the DNA double helix re-forms
Rate: roughly 40 nucleotides per second
Multiple RNA polymerase molecules can transcribe the same gene simultaneously, increasing output
Termination
In bacteria: the polymerase transcribes through a terminator sequence in the DNA. The resulting RNA sequence causes the polymerase to detach and release the transcript. No further processing is needed before translation.
In eukaryotes: RNA polymerase II transcribes past a polyadenylation signal sequence (AAUAAA in the pre-mRNA). Proteins bind this signal and cut the pre-mRNA free about 10 to 35 nucleotides downstream. The polymerase continues transcribing briefly; enzymes degrade the remaining RNA from its exposed 5' end until they catch up to the polymerase, which then dissociates.
5' Cap
A modified guanine nucleotide is added to the 5' end after the first 20 to 40 nucleotides are transcribed
Functions: protects mRNA from degradation, facilitates nuclear export, helps ribosomes attach
Poly-A Tail
After the pre-mRNA is cleaved at the AAUAAA signal, an enzyme adds 50 to 250 adenine nucleotides to the 3' end
Functions: same as the 5' cap (protection, export, ribosome attachment)
Untranslated Regions (UTRs)
Present at both the 5' and 3' ends of the mature mRNA
Not translated into protein, but serve functions such as ribosome binding
The average primary transcript is roughly 27,000 nucleotides, but only about 1,200 are needed to code for a typical 400-amino-acid protein
The extra length comes from introns: non-coding stretches interspersed between the coding exons
During RNA splicing, introns are removed and exons are joined into a continuous coding sequence
The spliceosome (a large complex of proteins and small RNAs called snRNPs) carries out splicing:
Small RNAs in the spliceosome recognise splice-site sequences at intron boundaries
The intron is cut out and degraded
The flanking exons are joined together
Some RNA molecules can act as enzymes (ribozymes)
In certain organisms, intron RNA can catalyse its own excision without proteins
Three properties of RNA enable this catalytic ability:
Single-stranded RNA can fold into complex 3D shapes (required for catalysis)
Some RNA bases have functional groups that can participate in chemical reactions
RNA can hydrogen-bond with other nucleic acids, providing specificity
Alternative RNA splicing: different exon combinations from the same gene produce different mRNAs and different polypeptides. This means an organism can make far more proteins than it has genes.
Exon shuffling: introns increase the probability of crossing over between exons, potentially recombining functional protein domains into new combinations
Different exons often code for different protein domains (discrete structural/functional regions)
Exon shuffling can also occur between entirely different (nonallelic) genes
Students often think RNA polymerase needs a primer. It does not. Only DNA polymerase requires a primer.
Students often confuse the promoter with the start codon. The promoter is a DNA sequence where RNA polymerase binds; the start codon (AUG) is part of the mRNA and is relevant to translation, not transcription.
Students often think introns are "junk DNA" with no function. Some introns contain regulatory sequences, and their presence enables alternative splicing and exon shuffling, both of which have major functional and evolutionary consequences.
Students often assume the 5' cap and poly-A tail are translated into protein. They are not. Both sit in the untranslated regions and serve protective and regulatory functions.
⚠️ Know the three stages of transcription (initiation, elongation, termination) and how they differ between bacteria and eukaryotes.
⚠️ Be able to explain the role of each mRNA modification: 5' cap, poly-A tail, and splicing. Expect a question asking you to list their shared functions.
⚠️ Understand what introns and exons are, and be able to diagram the transition from pre-mRNA (with introns) to mature mRNA (exons only, with cap and tail).
⚠️ Alternative splicing is a frequent exam topic. Be ready to explain how one gene can produce multiple proteins.
⚠️ The TATA box and transcription factors are commonly tested for eukaryotic transcription initiation.
True or False: RNA polymerase II synthesises pre-mRNA in eukaryotes. (True.)
Fill in the blank: The ______ is a promoter DNA sequence about 25 nucleotides upstream of the transcription start point. (TATA box)
True or False: In bacteria, the mRNA transcript requires a 5' cap and poly-A tail before translation. (False. Bacterial mRNA is translated directly, with no processing.)
Fill in the blank: Non-coding sequences within a gene are called ______; coding sequences are called ______. (introns; exons)
True or False: Ribozymes are protein enzymes that catalyse RNA splicing. (False. Ribozymes are RNA molecules that function as enzymes.)
Q: Compare transcription termination in bacteria and eukaryotes.
A: In bacteria, the polymerase transcribes through a terminator sequence; the resulting RNA folds into a structure that causes the polymerase to detach and release the transcript, which is ready for translation immediately. In eukaryotes, RNA polymerase II transcribes past a polyadenylation signal (AAUAAA). Proteins recognise this signal, cut the pre-mRNA free, and the polymerase continues briefly until enzymes degrade the trailing RNA and force dissociation. The released pre-mRNA then undergoes further processing.
Q: List the three modifications made to eukaryotic pre-mRNA and state one shared function.
A: The three modifications are addition of a 5' cap, addition of a poly-A tail, and RNA splicing (removal of introns). A shared function of the cap and tail is protecting the mRNA from degradation by hydrolytic enzymes.
Q: A eukaryotic gene has five exons and four introns. How many different mRNA molecules could potentially be produced by alternative splicing?
A: Many. Each combination of exons that maintains the reading frame produces a different mRNA. Even with five exons, dozens of combinations are possible (the exact number depends on which exons are compatible). The key point is that alternative splicing allows one gene to produce multiple distinct polypeptides.
Q: Explain how the discovery of ribozymes changed our understanding of biological catalysis.
A: Before ribozymes were discovered, it was assumed that all biological catalysts were proteins. Ribozymes showed that RNA can also catalyse chemical reactions, including its own splicing. This has implications for theories about the origin of life (the "RNA world" hypothesis) and expands our understanding of what molecules can serve as enzymes.
Q: Why do eukaryotic genes have introns? Give one functional and one evolutionary reason.
A: Functionally, introns enable alternative splicing, allowing one gene to produce multiple protein variants. Evolutionarily, introns facilitate exon shuffling: crossovers within introns can recombine exons from different genes, potentially creating proteins with novel domain combinations.
Transcription connects back to DNA structure and base-pairing (Ch. 16) and forward to gene regulation (Ch. 18), where promoters and transcription factors play a central role in controlling which genes are expressed. RNA processing, particularly alternative splicing, also connects to development and cell differentiation (Ch. 18, 21), where the same genome produces different proteins in different cell types.
transcription, RNA polymerase, RNA polymerase II, promoter, terminator, transcription unit, TATA box, transcription factors, transcription initiation complex, elongation, termination, 5' cap, poly-A tail, polyadenylation, UTR, pre-mRNA, primary transcript, RNA processing, RNA splicing, intron, exon, spliceosome, snRNP, ribozyme, alternative splicing, exon shuffling, protein domains, bacteria vs eukaryotes transcription