Molecular Genetics: DNA, RNA and the Genetic Code

Molecular Genetics carries 2 of the 40 Botany questions, and they are the most number-dense two marks in the whole paper. One question is almost always a structural fact — the width of the helix, the base pairs per turn, which bases are purines, how many hydrogen bonds hold A to T — and the other is a piece of code arithmetic or a one-line statement about replication, the enzymes or the properties of the genetic code. No calculation here is longer than a division. Learn the numbers in the tables and the three classic experiments, and this unit pays two marks for very little work.

8.1 Composition, structure and functions of DNA and RNA

The evidence that DNA is the genetic material

By 1940 most biologists assumed the genetic material was protein, because proteins are made of twenty different building blocks and DNA of only four. Three experiments destroyed that assumption. Each is a favourite of the paper, and each is worth one sentence in your memory.

Griffith, 1928 — transformation. Frederick Griffith worked with Streptococcus pneumoniae, the pneumococcus. The S strain has a polysaccharide capsule, forms smooth colonies and kills a mouse; the R strain has no capsule, forms rough colonies and is harmless. Griffith found that mice injected with heat-killed S bacteria plus live R bacteria died, and live S bacteria could be recovered from their blood. Something released by the dead S cells had permanently changed the harmless R cells into virulent S cells. He called it the transforming principle — but he did not know what it was. Note the logic carefully: neither component alone kills; the mixture does. That is the evidence.

Avery, MacLeod and McCarty, 1944 — the principle is DNA. Oswald Avery's group purified the transforming principle from heat-killed S cells and then destroyed each class of molecule in turn. Digested with protease (protein destroyed) the extract still transformed. Digested with ribonuclease (RNA destroyed) it still transformed. Digested with deoxyribonuclease (DNA destroyed) transformation stopped completely. The conclusion was that the transforming principle is DNA.

Hershey and Chase, 1952 — the label goes with the DNA. Alfred Hershey and Martha Chase used the T2 bacteriophage, which is nothing but a DNA molecule inside a protein coat. They grew one batch of phage in medium containing ³⁵S, which is built into the sulphur-containing amino acids of protein but not into DNA, and another in ³²P, which is built into the phosphate backbone of DNA but not into protein. Each batch was allowed to infect E. coli. A kitchen blender then sheared the empty coats off the bacterial surface, and centrifugation packed the bacteria into a pellet. Almost all the ³²P was inside the pellet with the bacteria; almost all the ³⁵S stayed in the supernatant with the coats. Since the injected material directs the making of new phage, the genetic material is DNA.

The nucleotide: sugar, phosphate and base

DNA and RNA are polynucleotides — chains of nucleotides. One nucleotide has three parts joined by two bonds:

• a pentose sugar — 2-deoxy-D-ribose in DNA (C₅H₁₀O₄), D-ribose in RNA (C₅H₁₀O₅);
• a phosphate group attached to carbon 5′ of the sugar;
• a nitrogenous base attached to carbon 1′ of the sugar.

A nucleoside is the sugar plus the base, without the phosphate. Adding the phosphate to a nucleoside gives the nucleotide: adenosine → adenosine monophosphate (AMP). Successive nucleotides are joined when the 3′-OH of one sugar makes a phosphodiester bond with the 5′-phosphate of the next. The result is a sugar–phosphate backbone with the bases projecting from it, and the chain has a chemical direction: a free phosphate at the 5′ end and a free hydroxyl at the 3′ end. Sequences are always written 5′ → 3′.

The two families of bases are told apart by their rings:

Thymine is 5-methyluracil; RNA uses uracil in its place, and uracil lacks the methyl group. In double-stranded DNA the number of purines always equals the number of pyrimidines, because every purine is paired with a pyrimidine.

The Watson–Crick double helix

James Watson and Francis Crick published the model in Nature in 1953. They used three things: the X-ray diffraction photographs of Rosalind Franklin and Maurice Wilkins (Franklin's "Photograph 51" showed a helix), the Chargaff ratios, and the chemistry of the bases. The model has these features, and the paper asks for them one by one:

1. Two polynucleotide chains wind round a common axis in a right-handed helix.
2. The chains are antiparallel — one runs 5′ → 3′ upwards, the other 5′ → 3′ downwards.
3. The sugar–phosphate backbones lie outside, and the bases point inwards, paired in the middle.
4. Each base pair is one purine with one pyrimidine, held by hydrogen bonds: A pairs with T by two hydrogen bonds, G pairs with C by three.
5. The two strands are complementary: the sequence of one fixes the sequence of the other. This is what makes faithful copying possible.
6. The helix has a major groove and a minor groove, where proteins bind.

Other forms of DNA. B-DNA is the Watson–Crick form and the one in living cells. A-DNA is also right-handed but shorter and fatter (11 bp per turn) and forms in dehydrated samples. Z-DNA is left-handed, with a zigzag backbone (12 bp per turn). Prokaryotes usually carry one circular DNA molecule; eukaryotes carry linear chromosomes, in which the DNA is wound round histone octamers into nucleosomes and then coiled into chromatin (Chapter 7).

Chargaff's rule and its arithmetic

Erwin Chargaff analysed DNA from many species in 1950 and found, without knowing the structure, that the bases are not present in equal amounts but are present in fixed pairs:

• %A = %T and %G = %C, so A + G = T + C — purines equal pyrimidines.
• The A + T to G + C ratio differs from species to species, but is constant within a species.

Every Chargaff question is solved in two lines. If one base is given, subtract its partner, halve the remainder, and remember that the four percentages add to 100.

Functions of DNA

• Storage of information. The base sequence is the information; a gene is a length of DNA that specifies one RNA or protein.
• Replication. Before a cell divides the DNA copies itself semi-conservatively, so both daughters receive a complete set.
• Control of the cell. Through transcription into RNA and then translation into protein, DNA decides the enzymes a cell makes and therefore its structure and behaviour.
• Mutation and variation. Rare copying errors change the sequence and provide the raw material of evolution.
• Inheritance. The gametes carry half the DNA of a body cell, so the information passes from parent to offspring.

RNA: the three types and their roles

RNA differs from DNA in four ways that the paper tests directly: it has ribose in place of deoxyribose, uracil in place of thymine, it is usually single-stranded (though it folds back on itself), and it is shorter and far less stable — the 2′-OH group of ribose makes RNA easy to hydrolyse, especially in alkali. Its single strandedness and its exposed bases let it fold into shapes and act as a catalyst.

Small nuclear RNA (snRNA) helps splice introns out of mRNA; micro-RNA regulates translation. In some viruses — tobacco mosaic virus, poliovirus, influenza, HIV — RNA is itself the genetic material, which is the exception to "DNA is the genetic material".

8.2 DNA replication; the central dogma; the genetic code

Semi-conservative replication

Three models were possible. In conservative replication the parent double helix stays intact and an entirely new one is made. In semi-conservative replication the two strands separate and each acts as a template, so every daughter molecule has one old strand and one new one. In dispersive replication the old and new material is broken into pieces and mixed.

Meselson and Stahl, 1958 settled it with E. coli and a heavy isotope of nitrogen. Bacteria were grown for many generations in ¹⁵N (as ¹⁵NH₄Cl), so all their DNA was heavy. They were then transferred to ordinary ¹⁴N medium, and samples were taken after each division. DNA was extracted and spun in a caesium chloride density gradient, where DNA bands at the position matching its density.

• Before the transfer: one heavy band at the bottom.
• After one division: one band of intermediate (hybrid) density, in the middle. This rules out conservative replication, which would have given two bands, one heavy and one light.
• After two divisions: two bands in equal amounts, one hybrid and one light. This rules out dispersive replication, which would have given a single band drifting upwards.

Only semi-conservative replication predicts exactly this. The same result was shown in plant roots by Taylor, Woods and Hughes (1957) using tritiated thymidine in Vicia faba.

The machinery of replication

Replication starts at an origin (one in a bacterial chromosome, many in a eukaryotic chromosome), and proceeds bidirectionally, so a replication bubble opens with a fork at each end. In eukaryotes it happens in the S phase of the cell cycle. Every enzyme has one job, and the paper asks "which enzyme does this?":

Leading and lagging strands

Because the two parental strands are antiparallel, and because DNA polymerase can only work 5′ → 3′, the two new strands cannot be made the same way.

• The leading strand is made continuously, in the same direction as the fork is moving, from a single primer.
• The lagging strand is made discontinuously, in short pieces that grow away from the fork, each started by its own primer. These pieces are the Okazaki fragments — about 100–200 nucleotides long in eukaryotes and 1000–2000 in bacteria. DNA polymerase I replaces their RNA primers and DNA ligase joins them into one strand.

So each fork produces one continuous and one fragmented strand. The lagging strand is the slower and more complicated of the two, and it is where the primers and the ligase are needed.

Transcription in outline

Transcription is the making of an RNA copy of one gene. RNA polymerase binds a promoter just upstream of the gene, unwinds a short stretch, and builds RNA 5′ → 3′ by reading the template strand in the 3′ → 5′ direction. The other strand, the coding (sense) strand, has the same sequence as the RNA except that T replaces U. No primer is needed, and only one strand of the gene is copied. The enzyme stops at a terminator.

In eukaryotes there are three polymerases: RNA polymerase I makes the large rRNA, RNA polymerase II makes mRNA, and RNA polymerase III makes tRNA and 5S rRNA. The mRNA is then processed: a 5′ cap of 7-methylguanosine, a 3′ poly-A tail, and splicing out of introns by the spliceosome.

The central dogma

Francis Crick stated the central dogma in 1958: information flows DNA → RNA → protein. Replication is DNA → DNA; transcription is DNA → RNA; translation is RNA → protein. The transfer is one-way in cells — protein sequence does not flow back into nucleic acid.

There are two real exceptions, and the paper likes them:

• Reverse transcription, RNA → DNA, catalysed by reverse transcriptase in retroviruses such as HIV. It is the basis of the enzyme used in the laboratory to make cDNA from mRNA.
• RNA replication, RNA → RNA, in some RNA viruses.

The genetic code

The code was broken between 1961 and 1966. Nirenberg and Matthaei made a synthetic mRNA of only uracil (poly-U) and got a polypeptide of only phenylalanine, so UUU codes for Phe. Khorana then made RNAs of known repeating sequence, and within five years all 64 codons had been assigned.

The wobble hypothesis (Crick, 1966) explains why a cell needs fewer than 61 tRNAs: the pairing between the third base of the codon and the first base of the anticodon is loose, so one tRNA can read several codons that differ only in that position. The first two bases pair strictly.

Translation

Translation turns the base sequence of mRNA into the amino acid sequence of a polypeptide. It happens on the ribosome — 70S (50S + 30S) in prokaryotes, 80S (60S + 40S) in eukaryotes — and it reads the mRNA 5′ → 3′.

1. Activation. Each amino acid is joined to its tRNA by an aminoacyl-tRNA synthetase, using ATP. The amino acid is attached to the CCA-3′ end.
2. Initiation. The small subunit binds the mRNA, the initiator tRNA carrying methionine pairs with the AUG start codon, and the large subunit joins. This tRNA sits in the P site.
3. Elongation. The next codon lies in the A site; the matching aminoacyl-tRNA binds; peptidyl transferase — the rRNA of the large subunit — forms the peptide bond; then the ribosome translocates one codon along, so the tRNA that has given up its amino acid moves to the E (exit) site and leaves.
4. Termination. A stop codon enters the A site, a release factor binds, and the polypeptide, mRNA and ribosomal subunits separate.

Several ribosomes may travel along one mRNA at once, forming a polysome, so many copies of the protein are made from one message.

Formula and fact sheet

Practice questions

Set A — Recall (23 questions)

1. Griffith's experiment with Streptococcus pneumoniae showed thataDNA is a double helixbheat-killed virulent bacteria can transform live avirulent onescprotein is the genetic materialdbacteria divide by binary fission
2. In the Avery–MacLeod–McCarty experiment, transformation of R into S pneumococci stopped when the extract was treated withadeoxyribonucleasebproteasecribonucleasedamylase
3. Hershey and Chase labelled the DNA of the T2 phage witha³⁵Sb¹⁵Nc³²Pd³H
4. Which pair of bases are purines?aadenine and thyminebcytosine and guaninecthymine and uracildadenine and guanine
5. A nucleoside consists ofaa sugar and a phosphateba sugar and a baseca base and a phosphatedtwo sugars and a base
6. Two nucleotides in one polynucleotide strand are joined byaa hydrogen bondba glycosidic bondca phosphodiester bondda peptide bond
7. The diameter of the DNA double helix isa2 nmb0.34 nmc3.4 nmd20 nm
8. One complete turn of B-DNA containsa10 base pairs and is 0.34 nm longb20 base pairs and is 3.4 nm longc34 base pairs and is 2 nm longd10 base pairs and is 3.4 nm long
9. In DNA, adenine is joined to thymine byaone hydrogen bondbthree hydrogen bondsctwo hydrogen bondsda covalent bond
10. Chargaff's rule for double-stranded DNA states thataA + T = G + CbA = T and G = CcA = G and T = CdA + G = 2(T + C)
11. The X-ray diffraction photographs used by Watson and Crick were made byaRosalind FranklinbErwin ChargaffcFrederick GriffithdOswald Avery
12. The most abundant RNA in a cell isamRNAbtRNAcrRNAdsnRNA
13. The three bases of tRNA that are complementary to a codon are calledacodonsbexonscintronsdanticodons
14. Which of the following is a stop codon?aAUGbUGAcUGGdGUG
15. The start codon AUG also codes foravalinebleucinecmethioninedtryptophan
16. The total number of codons in the genetic code isa64b61c20d128
17. Meselson and Stahl used which isotope to follow DNA replication?a³²Pb¹⁵Nc³⁵Sd¹⁴C
18. Okazaki fragments are joined together byahelicasebprimasecDNA polymerase IIIdDNA ligase
19. The enzyme that unwinds the double helix at the replication fork isaligasebprimasechelicasedtopoisomerase
20. The central dogma of molecular biology isaDNA → RNA → proteinbprotein → RNA → DNAcRNA → DNA → proteindDNA → protein → RNA
21. Reverse transcriptase synthesisesaRNA from a DNA templatebprotein from mRNAcDNA from a DNA templatedDNA from an RNA template
22. In eukaryotes, mRNA is made byaRNA polymerase IbRNA polymerase IIcRNA polymerase IIIdDNA polymerase I
23. The 5′ end of a polynucleotide chain carriesaa free hydroxyl groupban amino groupca free phosphate groupda nitrogenous base

Set B — Understanding (14 questions)

1. DNA is a better long-term store of information than RNA becauseaDNA contains uracil, which resists hydrolysisbDNA is single-stranded and therefore shortercDNA lacks the 2′-OH group, so it is chemically more stabledDNA is found only in the nucleus
2. A DNA sample with a high GC content has a higher melting temperature becauseaeach GC pair is held by three hydrogen bonds instead of twobGC pairs are joined by covalent bondscguanine and cytosine are larger basesdGC pairs lie on the outside of the helix
3. The Meselson–Stahl result after one generation in ¹⁴N medium ruled out conservative replication becauseatwo bands appeared, one heavy and one lightball the DNA remained heavycthe DNA was completely degradedda single band of intermediate density appeared
4. The lagging strand is made in short fragments becauseaDNA polymerase can synthesise only in the 3′ → 5′ directionbDNA polymerase adds only to a 3′-OH end, opposite this templatecthe lagging strand has no primer at alldligase can join only short pieces
5. DNA polymerase cannot start a new chain on its own becauseait needs a supply of ATPbit works only on single-stranded DNAcit can add nucleotides only to an existing free 3′-OH groupdit is inhibited by helicase
6. "The genetic code is degenerate" means thatamore than one codon can specify the same amino acidbone codon can specify more than one amino acidcsome bases are never useddthe code differs from species to species
7. A base substitution in the third position of a codon often does not change the protein becauseathe third base is not read by the ribosomebcode degeneracy lets the new codon specify the same amino acidcthe third base is removed during splicingdthe code is overlapping
8. A single base deletion usually alters the whole protein, whereas a substitution alters at most one amino acid, becauseadeletions always remove more materialba deletion removes a whole codoncsubstitutions are repaired and deletions are notda deletion shifts the reading frame of every downstream codon
9. tRNA is called an adaptor molecule becauseait carries the message out of the nucleusbit forms the structural core of the ribosomecone end binds an amino acid while its anticodon reads the codondit joins Okazaki fragments
10. One tRNA can read more than one codon for the same amino acid becauseaits anticodon is four bases longbthe 3rd codon base wobbles with the 1st base of the anticodoncthe ribosome corrects the pairingdthe code is ambiguous
11. RNA is more easily hydrolysed than DNA, especially in alkali, becauseaits ribose carries a 2′-OH groupbit contains uracilcit is single-strandeddit is much shorter
12. In the Hershey–Chase experiment the blender was used toabreak open the bacteriabrelease the DNA from the phagecmix the two isotopes evenlydshear the empty phage coats off the bacterial surface
13. Two cells of the same organism make different proteins mainly becauseathey contain different genesbdifferent genes are expressed in themctheir DNA contains different basesdtheir ribosomes are different
14. The genetic code is described as non-overlapping becauseano base is used twice anywhere in the mRNAbthe codons are separated by punctuationceach base is read in one codon only, and the next codon follows at oncedno two codons share an amino acid

Set C — Application (9 questions)

1. A double-stranded DNA sample contains 20 % cytosine. The percentage of adenine isa20b40c60d30
2. A DNA fragment of 100 base pairs contains 60 GC pairs. The total number of hydrogen bonds holding the two strands together isa200b260c300d240
3. The haploid human genome contains 3.2 × 10⁹ base pairs. The total length of DNA in one diploid somatic cell is abouta2.2 mb1.1 mc22 md0.22 m
4. A polypeptide contains 90 amino acids. The minimum number of nucleotides in the coding region of its mRNA, including the stop codon, isa90b180c273d270
5. An mRNA reads 5′-AUG CCU AAG UGA-3′. The polypeptide formed from it containsafour amino acidsbtwo amino acidscone amino aciddthree amino acids
6. The template strand of a gene reads 3′-TAC GGA TTC-5′. The mRNA transcribed from it isa5′-ATG CCT TTC-3′b5′-AUG CCU AAG-3′c5′-AUG GGA UUC-3′d5′-UAC GGA UUC-3′
7. A DNA molecule is 1.36 mm long. The number of complete turns of the helix it contains isa4 × 10³b4 × 10⁶c4 × 10⁵d4 × 10⁴
8. An E. coli chromosome has 4.6 × 10⁶ base pairs. Replication starts at one origin and each fork moves at 1000 nucleotides per second. The time taken is abouta38 minutesb77 minutesc19 minutesd230 minutes
9. A DNA sample contains 30 % guanine. The percentage of thymine in it isa30b20c40d70