The genetic code

A codon is a group of three nucleotides in mRNA. Four bases can make (4^3 = 64) codons: 61 sense codons specify the 20 standard amino acids, and three are stop signals.

AUG is the usual start codon. It specifies methionine; bacterial initiation uses N-formylmethionine (fMet). The stop codons are UAA, UAG, and UGA. A stop codon does not add an amino acid. A release factor recognizes it.

Example:

5′-AUG UUU GGC UAA-3′ → Met–Phe–Gly–STOP

The product contains three amino acids. UGA can be recoded as selenocysteine in the correct sequence context with specialized machinery, so “universal” and “always stop” are useful rules, not exception-free laws.

Codon, anticodon, and wobble

tRNA is the adaptor between nucleic acid and protein. Its anticodon recognizes the mRNA codon. Its conserved 3′-CCA end carries the amino acid on the terminal adenosine.

Codon and anticodon pair antiparallel:

• mRNA codon: 5′-AUG-3′
• Paired anticodon: 3′-UAC-5′
• The same anticodon written conventionally 5′→3′: 5′-CAU-3′

Always inspect the printed direction before choosing an anticodon. Complementing AUG to UAC without checking direction is the common trap.

The first two codon positions usually demand standard Watson–Crick geometry. The third codon base is more flexible. This is wobble, corresponding to the first base at the 5′ end of the anticodon. Wobble lets one tRNA recognize several synonymous codons and reduces the number of distinct tRNAs a cell needs.

An anticodon 5′-IGC-3′ can recognize 5′-GCU-3′, 5′-GCC-3′, and 5′-GCA-3′. The code has not changed; the pairing mechanism is flexible.

Degeneracy and wobble are not synonyms. Degeneracy is the fact that several codons encode the same amino acid. Wobble is how one anticodon can physically recognize several of those codons.

Charging the tRNA

Before a tRNA enters translation, an aminoacyl-tRNA synthetase (aaRS) attaches the correct amino acid. Charging is a separate accuracy checkpoint from ribosomal decoding.

1. Amino acid + ATP → aminoacyl-AMP + PPi
2. Aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

ATP is converted to AMP, and PPi is hydrolyzed. That costs the equivalent of two high-energy phosphate bonds. The amino acid’s carboxyl group forms an ester bond to a hydroxyl on the terminal A of the 3′-CCA end. Its amino group remains free for peptide-bond formation.

Many synthetases have an editing site that removes an incorrectly activated amino acid or hydrolyzes a mischarged tRNA. This matters because the ribosome mainly checks codon–anticodon pairing. If the correct anticodon carries the wrong amino acid, the ribosome may accept it and put the wrong residue into the protein.

Ribosomes and the A, P, and E sites

The small subunit decodes mRNA. The large subunit contains the peptidyl-transferase center.

S means Svedberg sedimentation coefficient. It depends on size, shape, and density and is not additive: 30S + 50S makes 70S, not 80S.

The three sites are easiest as one sentence: A accepts, P carries the peptide, E exits.

• A site: incoming aminoacyl-tRNA
• P site: tRNA carrying the growing polypeptide
• E site: deacylated tRNA before departure

In bacteria, 16S rRNA belongs to the 30S decoding subunit. 23S rRNA belongs to the 50S subunit and performs the central peptidyl-transferase chemistry. The ribosome is therefore a ribozyme.

Initiation fixes the reading frame

Initiation puts the start codon in the correct position and places the special initiator tRNA directly in the P site.

Elongation: delivery, bond, movement

The cycle must stay in this order:

1. Delivery and decoding. Bacterial EF-Tu–GTP carries an aminoacyl-tRNA to A. Correct pairing triggers GTP hydrolysis and accommodation. EF-Ts regenerates EF-Tu–GTP.
2. Peptide-bond formation. The amino group on the A-site amino acid attacks the ester-linked peptide on the P-site tRNA. The peptide transfers to the A-site tRNA. Bacterial 23S rRNA catalyzes the reaction.
3. Translocation. EF-G–GTP moves the ribosome one codon. Peptidyl-tRNA moves A→P; empty tRNA moves P→E and exits. A is free again.

Each elongation cycle uses two GTP in this simplified accounting: one for EF-Tu selection and one for EF-G translocation. This is in addition to the ATP→AMP cost of charging.

Termination and recycling

Termination begins when UAA, UAG, or UGA reaches A. There is no ordinary charged tRNA for stop. A release factor enters, and water hydrolyzes the ester bond between the completed chain and the P-site tRNA.

After release, bacterial RRF, EF-G, and IF-3 recycle the complex. Eukaryotes use ABCE1 and additional factors.

A premature termination codon can trigger nonsense-mediated mRNA decay (NMD). NMD lowers the amount of an mRNA that could produce a truncated protein. It is context-dependent and is an mRNA surveillance pathway, not ordinary termination.

Translation errors, variants, and laboratory evidence

A translation error affects a particular protein molecule without necessarily changing DNA or mRNA. A genetic variant changes the encoded sequence and can recur in every transcript or protein produced from that allele.

One method rarely answers every clinical question:

For a premature stop, separate three questions: confirm the DNA variant; test whether the RNA is spliced differently or degraded by NMD; then determine whether protein is absent, shortened, unstable, or inactive.

Say these without looking

• The ribosome reads mRNA 5′→3′; protein grows N→C.
• AUG starts. UAA, UAG, and UGA stop. Bacterial initiator amino acid is fMet.
• Wobble is the third codon base and first 5′ anticodon base. Degeneracy is not wobble.
• Aminoacyl-tRNA synthetase charges 3′-CCA. ATP→AMP costs two high-energy bond equivalents.
• 70S = 30S + 50S; 80S = 40S + 60S. S values are not additive.
• 16S decodes; 23S catalyzes peptide-bond formation.
• A accepts, P holds peptide, E exits. The initiator tRNA starts in P.
• Shine–Dalgarno is bacterial. Cap scanning and Kozak context are eukaryotic.
• EF-Tu delivers, EF-Ts reloads EF-Tu, EF-G translocates.
• Peptide bond first, translocation second.
• RF1 recognizes UAA/UAG; RF2 recognizes UAA/UGA; eRF1 recognizes all three.
• Missense substitutes, nonsense stops, frameshift changes the downstream frame.

Previous: Transcription and RNA processing. All lessons: MB(ASCP) study notes.

Check yourself

Ten original questions. Answer all of them before checking; the explanation appears with the key.

Sources and figure note

All three figures are original SVG study schematics created for this page. The primary lesson source is the supplied MB(ASCP) Lesson 4 — Genetic Code and Translation PDF, which identifies Lela Buckingham’s Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications, Chapter 1 (especially printed pp. 45–51 and Figures 1.38–1.41), as its main textbook basis. Eukaryotic initiation, factor equivalents, NMD, and diagnostic-method comparisons are supplementary exam context. Use the current ASCP Board of Certification content guideline to confirm the scope for your examination date.

What the polymerase reads

RNA polymerase reads the template strand 3′→5′ and writes RNA 5′→3′. It does not need a primer. The new RNA uses uracil where DNA would have used thymine.

The other DNA strand is the coding strand, also called the sense strand. Its sequence matches the RNA, with T standing where the RNA has U. The polymerase does not copy the coding strand. People get this backward because the coding strand “looks like” the product. It looks like the product because the template was its complement.

Read a short example as a habit:

The RNA is complementary to the template and identical to the coding strand except at U. If an item gives you the template and asks for the RNA, write the complement and flip the direction so the answer runs 5′→3′.

Three stages, and where bacteria and eukaryotes differ

A promoter decides where transcription starts and how eager the start is. It is DNA. It is not transcribed into the body of a bacterial mRNA as a coding sequence, and it is not the same thing as the start codon.

In bacteria the core RNA polymerase is α2ββ′ω. The sigma subunit is what recognizes the promoter, principally the −10 box (TATAAT) and the −35 box (TTGACA). Sigma leaves after initiation. The core enzyme elongates without it.

Bacterial termination comes in two versions. Intrinsic, or rho-independent, termination is an RNA hairpin rich in G–C, followed by a run of U. The weak A–U hybrid lets the chain fall off. Rho-dependent termination uses the rho protein, which loads on a C-rich rut site in the RNA and catches the polymerase.

Eukaryotes split the work among three nuclear polymerases:

The memory line is Pol I for most rRNA, Pol II for pre-mRNA, Pol III for tRNA and 5S. The item that catches people is 5S rRNA. It is ribosomal, but it is a Pol III product. U6 is the snRNA exception in the other direction: most snRNAs are Pol II, and U6 is Pol III.

Eukaryotic promoters are more varied. Many Pol II promoters have a TATA box near −25, recognized by TBP within TFIID. Others rely on an initiator, a CAAT box, or GC boxes. “Every eukaryotic gene has a TATA box” is false.

The RNA types worth naming

You do not need a catalog of every lncRNA. You need to know that a transcript can be functional without becoming a protein, and that mRNA, tRNA, and rRNA are three different answers.

What happens to eukaryotic pre-mRNA

Bacteria often translate an mRNA while it is still being made, and a bacterial mRNA does not receive a 5′ cap or a long poly-A tail of the eukaryotic kind. Eukaryotic protein-coding genes produce a pre-mRNA that is processed, mostly while polymerase II is still elongating. Phosphorylation of the Pol II C-terminal domain is what couples the polymerase to the processing machinery. The three events are the cap, splicing, and cleavage plus polyadenylation.

Reverse transcription, said so the abbreviations stop colliding

Reverse transcriptase reads RNA 3′→5′ and writes DNA 5′→3′. Unlike RNA polymerase, it needs a primer. For mRNA the primer is often oligo(dT), which pairs with the poly-A tail. Random primers are used when the RNA has no tail, or when the goal is to copy more than the 3′ end. Retroviral reverse transcriptase also has an RNase H activity that degrades the RNA half of the hybrid.

In the clinical lab:

• PCR starts from DNA.
• RT-PCR starts from RNA, makes complementary DNA with reverse transcriptase, and then runs PCR.
• qPCR, or real-time PCR, is a way of watching product accumulate, with a dye or a probe. It says nothing by itself about whether the input was RNA.
• RT-qPCR is both: reverse transcription, then real-time detection.

“RT” in a method name is the abbreviation that causes the miss. On these notes, RT means reverse transcription. Real-time is written out, or written as qPCR.

Say these without looking

• RNA polymerase reads the template 3′→5′ and writes 5′→3′. It does not need a primer.
• The coding strand matches the RNA, with T where RNA has U.
• Bacteria: sigma reads −10 and −35. Intrinsic termination is a hairpin plus a U run. Rho termination uses the rho protein.
• Pol I: 28S, 18S, 5.8S. Pol II: pre-mRNA. Pol III: tRNA, 5S rRNA, U6.
• The cap is 7-methylguanosine on a 5′–5′ link.
• AAUAAA is the common polyadenylation signal. The tail is added, not transcribed.
• Introns leave through the spliceosome. GU–AG and a branch-point adenine define the usual nuclear intron. Exons stay.
• Reverse transcriptase copies RNA into DNA and needs a primer. RT-PCR is that step plus PCR. qPCR is the detection format.

Previous: DNA replication. Next: Genetic code and translation. All lessons: MB(ASCP) study notes.

Check yourself

Where this sits

Both figures are original schematics. Promoters, the three nuclear polymerases, the cap, the spliceosome, and polyadenylation follow the account in Alberts and colleagues, Molecular Biology of the Cell. The clinical reading of RT-PCR versus real-time PCR follows Buckingham, Molecular Diagnostics. Polymerase product lists are the standard board assignments, including the two exceptions worth memorizing: 5S rRNA and U6 snRNA are RNA polymerase III products.

One old strand, one new strand

Replication is semiconservative. The parental strands separate, and each is the template for a new partner. Each daughter duplex therefore contains one parental strand and one new strand.

Meselson and Stahl showed this in E. coli by labeling DNA with heavy nitrogen (15N) and then shifting the cells to light nitrogen (14N). After exactly one generation in light medium, the DNA formed a single band of intermediate density. After exactly two generations, half of the DNA was intermediate and half was light. A conservative model would have kept a fully heavy duplex and made a fully light one. The two-generation result is the one that separates semiconservative copying from the alternatives.

Who stands at the fork

Helicase versus topoisomerase is a standard pair of distractors. Helicase separates strands. Topoisomerase manages the twist in front of that separation. Ligase is the enzyme of the nick, not the enzyme of the primer.

Direction, primers, and the two new strands

DNA polymerase reads the template 3′→5′ and writes the new strand 5′→3′. It can add a nucleotide only onto a free 3′-OH. That is why a primer exists. In the cell the primer is RNA, made by primase. In PCR the primer is a synthetic DNA oligo, and heat does the unwinding that helicase does in the cell.

The two templates are antiparallel, and the polymerase still writes only 5′→3′. One new strand can grow continuously toward the moving fork. That is the leading strand. The other new strand is made as short pieces, Okazaki fragments, each of them still 5′→3′ and pointed away from the fork. That is the lagging strand. A common miss is to say the lagging strand is polymerized 3′→5′. It is not. The fragments face the other way, and ligase joins them later.

Approximate fragment length, the range items expect:

Maturation is the same story in both kingdoms, with different enzymes. Primase makes the RNA primer. Polymerase extends it. The RNA is removed and the gap is filled with DNA. Ligase seals the last nick. In E. coli, DNA polymerase I removes the primer with its 5′→3′ exonuclease and fills the gap. In eukaryotes, RNase H and FEN1 do the removal work, polymerase fills, and ligase seals.

Which polymerase, on an exam

Bacterial assignments:

Eukaryotic assignments used on exams:

In the cell these enzymes share a replisome, and the labor is a little more shared than a one-enzyme-one-strand sentence suggests. For the item in front of you, use the table. Pol α starts the chain. Pol ε leads. Pol δ lags. Pol γ is the mitochondrial enzyme. A choice that makes Pol α the enzyme that finishes the chromosome is swapping the starter for the replicase.

Proofreading is a direction

Replicative polymerases check the base they just added. If it is wrong, a 3′→5′ exonuclease cuts it back off and synthesis continues. That activity is proofreading. Mismatch repair is a later, separate system. It is not the same activity.

The trap is to swap the two exonuclease directions. Proofreading walks backward on the new strand. Primer removal chews forward from the 5′ side of the RNA.

The ends of a linear chromosome

Bacterial chromosomes are circular, so the fork can finish. Eukaryotic chromosomes are linear. On the lagging strand, removing the terminal RNA primer leaves a gap, and no polymerase can fill it, because there is no upstream 3′-OH to extend. That is the end-replication problem.

Telomerase solves the shortening. It is a reverse transcriptase that carries its own RNA template and extends the 3′ overhang with telomere repeats. Ordinary DNA synthesis then fills part of the complementary strand. The overhang remains, and it is part of how the end is protected. If an item says “RNA-dependent DNA polymerase at the chromosome end,” the enzyme is telomerase.

The same rules, inside a PCR tube

Taq polymerase, from Thermus aquaticus, survives the denaturation step. Conventional Taq has little 3′→5′ proofreading, so the copied product accumulates errors. When the goal is a low error rate — cloning, for example — the reaction uses a high-fidelity polymerase that does proofread. Taq also tends to leave a single unpaired A on the 3′ end of the product. That overhang is a cloning detail, and it is fair game on a techniques item.

Say these without looking

• Replication is semiconservative. After two generations in light nitrogen, expect half intermediate and half light DNA.
• Helicase unwinds. Topoisomerase relieves twist ahead of the fork. Primase makes RNA primers. Ligase seals nicks.
• Polymerase writes 5′→3′ and can start only from a 3′-OH.
• Leading strand: one continuous chain toward the fork. Lagging strand: Okazaki fragments, each still 5′→3′.
• E. coli Pol III replicates the chromosome. Pol I removes primers with a 5′→3′ exonuclease.
• Exam assignment: Pol α starts, Pol ε leads, Pol δ lags, Pol γ replicates mitochondrial DNA.
• Proofreading is 3′→5′. Primer removal by Pol I is 5′→3′.
• PCR uses heat and DNA primers. Taq does not proofread well.

Previous: Nucleic acid chemistry. Next: Transcription and RNA processing. All lessons: MB(ASCP) study notes.

Check yourself

Where this sits

The fork drawing is an original schematic. Strand assignments for the eukaryotic polymerases follow the account used in exams and in standard texts (Pol ε on the leading strand, Pol δ on the lagging strand), with the caveat that the replisome in vivo is more cooperative than the table. Telomerase as an RNA-dependent DNA polymerase at chromosome ends is the same textbook account, as in Alberts and colleagues, Molecular Biology of the Cell. PCR enzyme behavior is the clinical-lab version of those rules, as taught in Buckingham, Molecular Diagnostics.

Three pieces, two names

Every nucleotide has a nitrogenous base, a five-carbon sugar, and at least one phosphate. The sugar–phosphate chain is the backbone. The order of the bases is the information.

The base is tied to carbon 1′ of the sugar by a glycosidic bond. Purines use nitrogen N9. Pyrimidines use nitrogen N1. The phosphate sits on carbon 5′. The next nucleotide will be attached to the free OH on carbon 3′.

Naming is part of the question. Adenine is the base, adenosine is the ribonucleoside, and AMP is the nucleotide. The prefix deoxy- means the sugar is 2′-deoxyribose, so dATP is a DNA building block. Thymidine is the usual name for the deoxyribonucleoside of thymine. Uridine is the ribonucleoside of uracil.

Purines and pyrimidines

Purines have two fused rings. Pyrimidines have one ring.

The memory line is enough: purines are A and G (“pure As gold”). Everything else in this family — C, T, and U — is a pyrimidine.

A few substituents are worth being able to picture. Adenine carries an amino group. Guanine carries an amino group and a carbonyl. Thymine is uracil with a methyl group on carbon 5, which is why thymine is also called 5-methyluracil. That methyl group is the only difference the exam wants between T and U.

DNA and RNA

The 2′-OH is the whole stability story the board wants. Under alkaline conditions that hydroxyl can attack the neighboring phosphate and cut the chain. DNA has no 2′-OH, so it does not have this path. Folding, proteins, and RNases still decide how long an RNA actually lasts in a tube. The chemical point remains: RNA is the alkali-labile nucleic acid because of the 2′-OH.

DNA polymerases are given dNTPs. RNA polymerases are given NTPs. ATP is both an energy cofactor and an RNA building block. If a stem says “the polymerase incorporates ATP,” check whether the product is RNA before you call it a trick.

Two bonds, and which direction the strands run

Hydrogen bonds pair bases across the two strands. A phosphodiester bond links nucleotides along one strand, from the 3′ carbon of one sugar to the 5′ phosphate of the next.

G–C is the stronger pair because it has three hydrogen bonds. Base stacking along the strand stabilizes the duplex as well. The exam still treats “three versus two” as the short answer.

The strands of a duplex are antiparallel. One runs 5′→3′. Its partner runs 3′→5′. Sequence is written 5′ to 3′ unless the item says otherwise.

In double-stranded DNA, Chargaff’s rules follow from that pairing: the amount of A equals T, and the amount of G equals C. A single strand does not have to obey that equality. An item that applies Chargaff to one RNA strand is using the rule in the wrong place.

Melting temperature

Tm is the temperature at which half of the duplexes in a population are denatured, under the conditions you actually used. As the strands separate, absorbance at 260 nm rises. That rise is the hyperchromic effect, and it is how a melting curve is read.

What pushes Tm up, when the other variables stay put:

• More G–C pairs, because each contributes three hydrogen bonds and favorable stacking.
• A longer perfectly matched duplex.
• Higher salt. Monovalent cations shield the repulsion between the phosphate backbones.

What pushes Tm down:

• Mismatches.
• A shorter duplex.
• Denaturants such as formamide or urea, which is why hybridization buffers use them when the goal is a more stringent, or a more controlled, melt.

GC percent is a first estimate. Primer-design software uses nearest-neighbor parameters, because the neighbors of a pair change the stability of that pair. Two oligos with the same GC percent can still have different Tm values. For a board item that only gives GC percent, length, and “otherwise identical conditions,” choose the higher GC.

In the lab this shows up as soon as you set an annealing temperature. Primers that melt far apart, or an annealing step set far below the primer Tm, are the usual reasons a PCR becomes either weak or nonspecific. The chemistry lesson and the amplification lesson are the same fact seen from two sides.

Say these without looking

• Purines: A and G, two rings, glycosidic bond through N9.
• Pyrimidines: C, T, and U, one ring, glycosidic bond through N1.
• Nucleoside = base + sugar. Nucleotide = nucleoside + phosphate.
• DNA sugar is 2′-deoxyribose and the unique base is T. RNA sugar is ribose and the unique base is U.
• The 2′-OH makes RNA alkali-labile.
• A–T and A–U have two hydrogen bonds. G–C has three.
• Phosphodiester bonds run along a strand. Hydrogen bonds pair the strands. The strands are antiparallel.
• Tm is 50% denaturation. Higher GC, greater length, and higher salt raise it. Mismatches and formamide lower it.

Next: DNA replication. All lessons: MB(ASCP) study notes.

Check yourself

Ten original questions. Answer all of them, then check. A miss opens a one-line reason.

Where this sits

Figures on this page are original schematics. The chemistry is the standard account used for clinical molecular boards: nucleotide structure, pairing, and melting behavior as treated in Alberts and colleagues, Molecular Biology of the Cell, and in the nucleic-acid chapters of Buckingham, Molecular Diagnostics. Use the current ASCP Board of Certification content guideline for MB(ASCP) and MB(ASCPi) to decide what else is in scope for your sitting.