Gene expression and regulation · AP Biology

Chapter 6 · Gene expression and regulation

from DNA to protein

A gene is a stretch of DNA whose sequence of bases spells out an RNA, and usually a protein. Expressing it takes two steps: transcription, DNA to RNA, and translation, RNA to protein.

The figure is a piece of the double helix, turning. Its two strands are held together by the base pairs in the middle, and the order of those bases is the information.

This chapter follows that information: how DNA is built (6.1) and copied (6.2), how a gene is read into RNA (6.3) and into protein (6.4), how cells decide which genes to read (6.5, 6.6), what happens when the sequence changes (6.7), and how a lab cuts, copies and sorts DNA (6.8).

6.1 · DNA and RNA

DNA and RNA structure

DNA, and in some viruses RNA, is the heritable information of a cell. Both are chains of nucleotides: a sugar, a phosphate and a nitrogenous base.

Sugars and phosphates alternate to form the backbone. Each strand has a direction: a 5′ end with a free phosphate and a 3′ end with a free –OH on the sugar. The two strands of DNA run opposite ways, antiparallel, and twist into a double helix.

The bases pair across the middle by hydrogen bonds: A with T (two bonds) and G with C (three). A and G are purines, with two rings; C, T and U are pyrimidines, with one. Every pair is one purine and one pyrimidine, so the helix keeps the same width (about 2 nm), turning once every 10.5 base pairs.

Chargaff’s rule. Because of the pairing, double-stranded DNA has A = T and G = C. If a sample is 30% A, it is 30% T, and the remaining 40% is split equally: 20% G and 20% C.

	DNA	RNA
sugar	deoxyribose	ribose
bases	A, T, G, C	A, U, G, C
strands	two, a double helix	usually one

Prokaryotes keep their DNA in one circular chromosome; eukaryotes in several linear chromosomes, wound on histone proteins in the nucleus. Both can carry plasmids: small, circular, double-stranded DNA outside the chromosome.

6.2 · Copying DNA

replication

Replication is semiconservative: each new double helix keeps one old strand and gains one new one, built on it as a template by base pairing.

At the replication fork, helicase unwinds the parent helix, breaking the hydrogen bonds between the bases. Topoisomerase, ahead of the fork, relaxes the twisting that unwinding causes.

DNA polymerase adds nucleotides only to the 3′ end of a strand, so a new strand grows only 5′→3′. And it cannot start a strand from nothing: it needs a short RNA primer (blue) to build on.

The two templates run opposite ways, so the two new strands (red) are made differently:

	leading strand	lagging strand
direction	toward the fork	away from the fork
made	continuously, one primer	in pieces, a primer each

The pieces of the lagging strand are Okazaki fragments. Their RNA primers are replaced with DNA, and DNA ligase seals the gaps between them into one continuous strand.

DNA polymerase also proofreads, so replication errors are rare, though never zero: the ones that remain are mutations (6.7).

6.3 · DNA to RNA

transcription and RNA processing

In transcription, RNA polymerase opens the DNA at a gene and builds an RNA copy of one strand.

It binds at the promoter, reads the template strand 3′→5′, and builds the RNA 5′→3′, pairing A with U, T with A, G with C and C with G. It stops at a terminator. The RNA has the sequence of the other, coding strand, with U for T.

coding strand 5′→3′	ATG TTC GGA AAA TGG TAA
template strand 3′→5′	TAC AAG CCT TTT ACC ATT
mRNA 5′→3′	AUG UUC GGA AAA UGG UAA

Three kinds of RNA take part in making a protein: mRNA carries the gene’s message; tRNA brings the amino acids; rRNA forms the core of the ribosome.

Processing. In eukaryotes the first transcript, the pre-mRNA, is modified in the nucleus before it leaves: a 5′ cap (a modified guanine nucleotide) and a poly-A tail are added, protecting it and helping it get translated, and the introns are spliced out while the exons are joined. The pre-mRNA below is 1530 nucleotides; its exons total 480.

By alternative splicing, one gene can yield different mRNAs, and so different proteins, depending on which exons are kept.

6.4 · RNA to protein

translation

In translation, a ribosome reads an mRNA three bases at a time, one codon per amino acid.

Initiation. The small subunit binds the mRNA and finds the start codon, AUG; the first tRNA, carrying methionine, pairs with it; the large subunit joins.

Elongation. Each tRNA has an anticodon that pairs with one codon, and carries that codon’s amino acid. A new tRNA enters the A site; the ribosome joins the growing chain to its amino acid by a peptide bond; the ribosome moves one codon toward the 3′ end, and the empty tRNA leaves from the E site.

Termination. At a stop codon (UAA, UAG, UGA) no tRNA pairs; a release factor frees the polypeptide.

mRNA	AUG	UUC	GGA	AAA	UGG	UAA
amino acid	Met	Phe	Gly	Lys	Trp	Stop

The code is read from the genetic code table, and it is nearly universal: the same codons mean the same amino acids in bacteria and in people, which is why a human gene can be expressed in a bacterium. It is also redundant: 61 codons for 20 amino acids.

In prokaryotes, ribosomes translate an mRNA while it is still being transcribed. Retroviruses such as HIV run the flow backward first: their reverse transcriptase copies the viral RNA into DNA, which joins the host’s genome.

6.5 · Switching genes on and off

regulation of gene expression

A cell does not express all of its genes all the time. Regulatory sequences in the DNA bind regulatory proteins, which turn transcription up or down.

The lac operon of E. coli is an operon: genes for one job (using lactose) under one promoter and one operator, transcribed into one mRNA. It is inducible, normally off:

condition	repressor	lacZYA
lactose absent	repressor on the operator	off
lactose present	allolactose holds the repressor off	on

The repressor, made by the separate gene lacI, sits on the operator and blocks RNA polymerase. When lactose is present, allolactose (made from it) binds the repressor and changes its shape; it falls off, and the genes are transcribed. (Transcription is strongest when glucose is also scarce.)

The trp operon is repressible, normally on. Its repressor is made inactive; when tryptophan is plentiful, tryptophan binds it, the repressor binds the operator, and the genes for making tryptophan turn off.

Epigenetic changes regulate genes without changing the sequence. Acetylation of histones loosens the DNA on them and allows transcription; methylation of DNA usually silences it. These marks are reversible, and can be passed on when cells divide.

6.6 · One genome, many cell types

gene expression and cell specialization

Nearly every cell in your body has the same genes. Cells differ because they express different ones.

A eukaryotic gene is turned on by transcription factors. General factors help RNA polymerase bind the promoter. Specific activators bind enhancers, which may lie thousands of bases away; the DNA bends so that the activator touches the machinery at the promoter, and transcription rises. Repressors bind and block it.

In the figure: the chromatin is condensed and silent; acetyl groups (red) loosen the histones; an activator (blue) binds the enhancer, the DNA loops, and mRNAs are made.

cell	actin	albumin	insulin	crystallin
liver	on	on	—	—
pancreas	on	—	on	—
lens	on	—	—	on

Each cell type has its own set of transcription factors, and so its own set of active genes and proteins. Genes that share a regulatory sequence respond to the same factor, so a single signal can switch on a whole program together.

Expression is also tuned after transcription: small RNAs (such as microRNAs) pair with particular mRNAs and block their translation or have them destroyed.

6.7 · When the sequence changes

mutations

A mutation is a change in the DNA sequence. It can change the protein a gene makes, and so the phenotype.

The figure mutates the mRNA AUG GAG UUC AAA UGG GCU UAA, which codes for Met-Glu-Phe-Lys-Trp-Ala:

mutation	codon	protein
silent	GAG → GAA	Met-Glu-Phe-Lys-Trp-Ala
missense	GAG → GUG	Met-Val-Phe-Lys-Trp-Ala
nonsense	AAA → UAA	Met-Glu-Phe (stop)
frameshift	UUC → CUU	Met-Glu-Leu-Gln-Met-Gly-Leu

A silent substitution changes a codon to another for the same amino acid. A missense substitution changes one amino acid: GAG → GUG, glutamate to valine, is the sickle-cell mutation in hemoglobin. A nonsense substitution makes a stop codon and cuts the protein short. An insertion or deletion of one base is a frameshift: every codon after it is read in the wrong frame.

Mutations arise from errors in replication or repair, and from radiation and chemicals. They are random with respect to what the organism needs; their effect may be harmful, neutral or beneficial depending on the environment. They are the original source of genetic variation.

Whole chromosomes change too: nondisjunction in meiosis gives a gamete an extra or missing chromosome, as in trisomy 21 (Down syndrome). Bacteria also gain genes sideways: transformation (taking up DNA), transduction (carried by a virus), conjugation (cell to cell) and transposition (genes moving within a genome).

6.8 · Reading and copying DNA

biotechnology

Four lab methods follow from what DNA is.

Gel electrophoresis sorts DNA by size. DNA is negative (its phosphates), so in an electric field it runs toward the + end; the gel holds back long pieces more than short ones, so the shortest travel farthest. A ladder of known sizes is run beside the samples. Distance falls steadily with the logarithm of size:

size (bp)	1500	1000	700	500	300	200	100
distance (mm)	10.7	16.0	20.6	25.0	31.7	37.0	46.0

A band 27.9 mm from the well lies between the 500 and 300 bp bands; reading it on the line gives about 400 bp. In the figure, the crime-scene sample’s bands match suspect 2’s.

PCR (polymerase chain reaction) copies a chosen stretch of DNA: heat to 95 °C to separate the strands, cool to about 55 °C so primers bind either side of the target, then 72 °C for a heat-stable DNA polymerase to extend them. Each cycle doubles the target: 5 cycles turn one copy into 32, and 30 cycles into about 1.07 billion.

Bacterial transformation puts a gene into bacteria on a plasmid, often with a gene for antibiotic resistance so that only transformed cells grow on a plate with the antibiotic.

DNA sequencing reads the order of the bases.