D. Molecular Genetics · Molecular GeneticsGene Expression I: Transcription
D3.3 D3.2 D2.1 D2.2About 70 minutes
Learning goals- I can state the central dogma and identify exceptions to it.
- I can identify the promoter, template strand, coding strand and terminator of a gene.
- I can describe initiation, elongation and termination of transcription in prokaryotes and eukaryotes.
- I can write the mRNA transcribed from a DNA sequence, with correct 5′ and 3′ ends.
- I can compare the three eukaryotic RNA polymerases and the roles of the RNAs they make.
Words to know
central dogma
The principle that genetic information flows from DNA to RNA to protein.
gene expression
The process by which the information in a gene is used to make a functional product, usually a protein.
transcription
Making an RNA copy of a gene, using one DNA strand as a template.
RNA polymerase
The enzyme that unwinds DNA and builds RNA 5′→3′ from a DNA template, without a primer.
promoter
A DNA sequence near the start of a gene where RNA polymerase (with helper proteins) binds.
template strand
The DNA strand that is read by RNA polymerase (also called the antisense strand).
coding strand
The DNA strand that is not read; its sequence matches the RNA (with T instead of U). Also called the sense strand.
sigma factor
In bacteria, a protein that helps RNA polymerase recognize the promoter.
TATA box
A promoter sequence (rich in T and A) about 25 to 30 base pairs upstream of the start site in many eukaryotic genes.
transcription factor
A protein that binds DNA and helps (or blocks) RNA polymerase from starting transcription.
terminator
A DNA sequence that signals the end of transcription.
From a recipe book you can't take home
Imagine a library cookbook that can never leave the reference room. To cook a dish you copy that one recipe onto a card and take the card to the kitchen. In a eukaryotic cell, the DNA stays in the nucleus; the "kitchen" (the ribosomes) is in the cytoplasm. Transcription makes the card: a short, disposable RNA copy of one gene. Translation (two lessons from now) uses the card to make a protein.
This lesson and the next three cover expectation D3.3: the steps of protein synthesis.
The central dogma
In 1958 Francis Crick summarized the flow of genetic information as DNA → RNA → protein. He called it the central dogma of molecular biology.
- Replication: DNA → DNA (copying the whole genome).
- Transcription: DNA → RNA (copying one gene).
- Translation: RNA → protein (decoding the RNA).
There are exceptions. Retroviruses such as HIV carry RNA genomes and use the enzyme reverse transcriptase to make DNA from RNA. Telomerase (from the replication lesson) is also a reverse transcriptase. Some viruses copy RNA directly into RNA. And many genes produce RNAs that are never translated (tRNA, rRNA, microRNA). But no known process copies a protein's sequence back into nucleic acid.
The parts of a gene
A gene is not just the part that codes for protein. Three regions matter for transcription:
- The promoter, just upstream (toward the 5′ side of the coding strand), where RNA polymerase binds.
- The transcribed region, starting at the start site (+1).
- The terminator, which signals the end.
Only one DNA strand is copied for a given gene: the template strand. RNA polymerase reads it 3′→5′ and builds RNA 5′→3′. The RNA is therefore complementary to the template and has the same sequence as the other strand, the coding strand, except that it has U wherever the coding strand has T. By convention, gene sequences are usually written as the coding strand, 5′→3′.
Which strand is the template can differ from gene to gene on the same chromosome; it depends on which way the promoter faces.
Transcription in bacteria
Bacteria have a single kind of RNA polymerase.
1. Initiation. A protein called the sigma factor attaches to RNA polymerase and recognizes two short promoter sequences, centred about 10 and 35 base pairs upstream of the start site (the "−10" sequence, often close to TATAAT, and the "−35" sequence, often close to TTGACA). RNA polymerase binds and unwinds about a dozen base pairs, forming a small transcription bubble.
2. Elongation. RNA polymerase moves along the template and adds ribonucleoside triphosphates (ATP, UTP, GTP, CTP) to the 3′ end of the RNA, pairing A with U, T with A, G with C and C with G. Unlike DNA polymerase, RNA polymerase needs no primer. The sigma factor soon drops off. Behind the enzyme, the DNA re-forms its double helix.
3. Termination. In many bacterial genes, the terminator is copied into an RNA sequence that is rich in G and C and can pair with itself, forming a hairpin, followed by a run of U's. The hairpin makes the polymerase pause, and the weak A-U pairs let the RNA slip off. Other genes use a helper protein called rho to pull the RNA away.
Bacteria have no nucleus, so ribosomes start translating the mRNA while it is still being transcribed. Bacterial mRNAs often carry the code for several proteins in a row (you will see why in the lac operon lesson).
Transcription in eukaryotes
The principles are the same, but there is more control and more machinery.
Three RNA polymerases. Eukaryotic nuclei have three:
- RNA polymerase I makes most ribosomal RNA (rRNA).
- RNA polymerase II makes messenger RNA (mRNA) for protein-coding genes, plus some small RNAs.
- RNA polymerase III makes transfer RNA (tRNA) and one small rRNA.
Careful: these Roman numerals are not the same as bacterial DNA polymerases I, II and III from the replication lesson. Always say which kind of polymerase you mean.
Initiation. RNA polymerase II cannot recognize a promoter by itself. General transcription factors do that. In many genes, a factor binds the TATA box about 25 to 30 base pairs upstream of the start site; other factors and RNA polymerase II then assemble into a transcription initiation complex. In the lesson on controlling genes in eukaryotes you will see how other proteins, bound to DNA far away, turn this complex up or down.
Elongation works as in bacteria.
Termination. Near the end of the gene, RNA polymerase II copies a signal sequence, AAUAAA, into the RNA. Proteins recognize it and cut the RNA a short distance downstream; the polymerase soon falls off the DNA.
The RNA made this way is a pre-mRNA (primary transcript). Before it can leave the nucleus it must be processed. That is the next lesson.
| Feature | Prokaryotes | Eukaryotes |
|---|
| RNA polymerases | one | three (I: rRNA; II: mRNA; III: tRNA) |
| Promoter recognition | sigma factor; −10 and −35 sequences | general transcription factors; often a TATA box |
| Location | cytoplasm (no nucleus) | nucleus |
| Coupled with translation? | yes, ribosomes start on growing mRNA | no, mRNA is processed and exported first |
| Genes per mRNA | often several (polycistronic) | usually one |
| Termination | hairpin + U's, or rho protein | after AAUAAA signal, RNA cut and released |
Worked exampleExample 1: From template strand to mRNA
The template strand of a short gene reads 3′-TACCGAATGCCT-5′. Write (a) the mRNA and (b) the coding strand, each 5′→3′.
- RNA is complementary and antiparallel to the template. Pair each base: T→A, A→U, C→G, G→C.
- Under 3′-TACCGAATGCCT-5′, the RNA reads 5′-AUGGCUUACGGA-3′ (its 5′ end lines up with the template's 3′ end, so no reversing is needed).
- The coding strand has the same sequence as the RNA with T for U: 5′-ATGGCTTACGGA-3′.
(a) mRNA 5′-AUGGCUUACGGA-3′; (b) coding strand 5′-ATGGCTTACGGA-3′.
Worked exampleExample 2: Which strand is the template?
A double-stranded DNA segment is shown below. An mRNA made from it begins 5′-AUGCCAG...-3′. Which strand was the template?
Top: 5′-ATGCCAGTTTGA-3′ Bottom: 3′-TACGGTCAAACT-5′
- The mRNA matches the strand with the same sequence (with U for T). The top strand reads ATGCCAG..., matching AUGCCAG...
- So the top strand is the coding strand.
- The bottom strand, 3′-TACGGTCAAACT-5′, is the template; it is read 3′→5′ from left to right.
The bottom strand is the template; the full mRNA is 5′-AUGCCAGUUUGA-3′.
Just for funRNA polymerase (like its cousin primase) is brave enough to start a new chain with no primer at all. DNA polymerase won't even get out of bed without one.
Hands-on activityTranscribe a "gene" with two colours
You need: two colours of paper or sticky notes, a marker, tape, and a long table.
- Make a 12-base double-stranded "gene": write the coding strand 5′-ATGAAAGGCTGA-3′ on one colour, one base per note, and line up the complementary template strand under it (3′-TACTTTCCGACT-5′). Add a "promoter" note on the left of the coding strand and a "terminator" note on the right.
- You are RNA polymerase. Start at the promoter. "Unwind" two or three base pairs by separating the notes slightly.
- On the second colour, write RNA nucleotides that pair with the template strand (remember U, not T) and tape them in a row below, adding each to the 3′ end.
- As you move right, push the DNA strands behind you back together.
- At the terminator, stop and release your RNA.
- Compare your RNA with the coding strand. What is the only difference?
What to look for: the RNA should read 5′-AUGAAAGGCUGA-3′, identical to the coding strand except U replaces T. The template strand is unchanged at the end: transcription copies information without using up the gene.
Your turn
1State the central dogma and give one exception.
DNA → RNA → protein. Exceptions: reverse transcription (RNA → DNA) by retroviruses such as HIV or by telomerase; RNA → RNA copying by some viruses.
2A template strand is 3′-TACGGCATTACT-5′. Write the mRNA 5′→3′.
3A coding strand is 5′-ATGTTCGGA-3′. Write the mRNA.
The mRNA has the coding strand's sequence with U for T: 5′-AUGUUCGGA-3′.
4What does the sigma factor do?
It helps bacterial RNA polymerase recognize and bind the promoter (the −10 and −35 sequences).
5Name the eukaryotic RNA polymerase that makes mRNA, and the one that makes tRNA.
mRNA: RNA polymerase II. tRNA: RNA polymerase III.
6Give two differences between DNA polymerase and RNA polymerase.
Any two: RNA polymerase needs no primer; it uses ribonucleotides (with U) instead of deoxyribonucleotides; it copies only one gene, not the whole genome; it unwinds DNA itself (no separate helicase); it does not proofread as thoroughly.
More practice: warm-up
1What base in RNA pairs with adenine in the template?
2In which direction is RNA built?
3Where does RNA polymerase first bind?
4Which strand has the same sequence as the mRNA (with T for U)?
5Where does transcription take place in a eukaryotic cell?
More practice: on your own
1Explain why the RNA has the same sequence as the coding strand even though it is made from the template strand.
The RNA is complementary to the template, and the coding strand is also complementary to the template. Two sequences complementary to the same strand must be identical (except that RNA uses U where DNA has T).
2Describe how a bacterial terminator stops transcription without a helper protein.
The terminator DNA is copied into RNA that is GC-rich and self-complementary, so it folds into a hairpin. The hairpin makes RNA polymerase pause; it is followed by a run of U's, which form weak A-U pairs with the template, so the RNA separates and is released.
3Why can bacteria translate an mRNA while it is still being transcribed, but eukaryotes cannot?
Bacteria have no nuclear envelope, so ribosomes can reach the growing mRNA right away. In eukaryotes, transcription happens in the nucleus and the mRNA must be processed and exported before ribosomes in the cytoplasm can use it.
4A toxin called alpha-amanitin, found in some poisonous mushrooms, strongly blocks RNA polymerase II. Which kind of RNA would stop being made, and why would this be so harmful?
mRNA would stop being made. Without new mRNA, cells cannot make new proteins, and the liver (which processes the toxin) is especially damaged. Cells depend on a steady supply of new mRNA.
5The TATA box is about 25 base pairs upstream of the start site. Why is its position as important as its sequence?
The transcription factor that binds the TATA box positions RNA polymerase II a fixed distance from it, so the position sets where transcription begins. Moving it would shift or prevent the start.
Challenge
1Two genes lie next to each other on the same chromosome. Gene X is transcribed left to right and gene Y right to left. Explain how this is possible and which strand each uses as its template.
Each gene uses whichever strand is oriented so that RNA polymerase reads it 3′→5′ in the direction its promoter points. Gene X (left to right) uses the strand whose 3′ end is on the left; gene Y (right to left) uses the other strand. The direction is set by the promoter's orientation.
2In an experiment, a researcher mixes purified bacterial RNA polymerase without sigma factor with DNA and nucleotides. RNA is made, but it starts at random places on both strands. Explain the result.
Without the sigma factor, RNA polymerase can still bind DNA and build RNA, but it cannot recognize promoters. So it starts at random sites on either strand instead of at the correct start sites. This shows sigma's role is promoter recognition.
3An mRNA has the sequence 5′-AUGGCAUGA-3′. (a) Write both strands of the gene that made it, with ends labelled. (b) Which is the template? (c) Is it possible to tell from the mRNA alone where the promoter was? Explain.
(a) Coding: 5′-ATGGCATGA-3′; template: 3′-TACCGTACT-5′. (b) The template is 3′-TACCGTACT-5′. (c) No. The promoter is upstream of the start site and is not transcribed, so its sequence is not in the mRNA. You only know it was on the 5′ side (upstream) of the coding strand.
Quiz
1. RNA polymerase reads the template strand in which direction?
2. The template strand of a gene is 3′-TACGAT-5′. The mRNA is:
3. Which statement correctly describes the coding strand?
4. In bacteria, which protein helps RNA polymerase recognize a promoter?
5. Which eukaryotic enzyme transcribes protein-coding genes into mRNA?
6. Which is an exception to the original central dogma?
Big idea (D3.3, D3.2): transcription copies one gene into RNA. RNA polymerase binds the promoter, reads the template strand 3′→5′, builds RNA 5′→3′, and stops at the terminator. The RNA matches the coding strand (with U for T).
How to help: Give a short DNA strand and ask, "If this is the template, what is the mRNA? If it's the coding strand, what is the mRNA?" Your teen should see that the two answers differ.
Common difficulties:
- Using T instead of U in RNA.
- Mixing up the template and coding strands.
- Confusing RNA polymerase I, II, III (eukaryotic transcription) with DNA polymerase I, II, III (bacterial replication).
- Thinking the promoter is part of the mRNA. It is not transcribed.