Transcription, RNA processing & translation

From DNA to protein in two steps

A gene's instructions are stored in DNA, but proteins are built elsewhere, on ribosomes. So the cell first makes an RNA copy of the gene. This copying step is transcription, and the copy that carries a protein recipe is messenger RNA (mRNA). Then ribosomes read the mRNA and build a chain of amino acids. That's translation.

RNA differs from DNA in three ways that matter here: its sugar is ribose instead of deoxyribose, it uses the base uracil (U) where DNA uses thymine (T), and it's usually single-stranded.

Transcription: template and coding strands

Only one of the two DNA strands is read for any given gene. The strand that RNA polymerase actually reads is the template strand. The new RNA is complementary to it, just as a new DNA strand is complementary to its template in replication.

The other strand is called the coding strand (or non-template strand). Because the mRNA is complementary to the template, and the coding strand is also complementary to the template, the mRNA has the same sequence as the coding strand, except that it has U wherever the coding strand has T. That's why gene sequences in databases are written as the coding strand: you can read the mRNA straight off it.

1. 1Initiation: RNA polymerase binds a DNA sequence called the promoter, just upstream of the gene. In bacteria a protein subunit called sigma (σ) recognises two short promoter sequences, about 10 and 35 bases upstream of the start. In eukaryotes, proteins called general transcription factors must assemble at the promoter (often at a sequence called the TATA box) before RNA polymerase II can bind.
2. 2Elongation: the polymerase unwinds a short stretch of DNA and adds RNA nucleotides that pair with the template. Like DNA polymerase, it adds only to a 3′ end, so RNA grows 5′→3′ while the template is read 3′→5′. Unlike DNA polymerase, it needs no primer.
3. 3Termination: at the end of the gene, a termination signal makes the polymerase let go of the DNA and release the RNA.

Eukaryotes process their RNA first

In a eukaryotic cell, transcription happens in the nucleus and translation in the cytoplasm, and the first RNA made, the pre-mRNA, is not yet ready to leave. Three changes turn it into mature mRNA.

• 5′ cap: a modified guanine nucleotide (7-methylguanosine) is attached to the 5′ end. It protects the mRNA from being chewed up by enzymes, and the ribosome's helper proteins recognise it when starting translation.
• Poly-A tail: a run of about 200 adenine nucleotides is added to the 3′ end. It also protects the mRNA from breakdown and helps it get exported from the nucleus.
• Splicing: most eukaryotic genes are interrupted by stretches that don't end up in the protein recipe, called introns. The parts that are kept are exons. A large RNA–protein machine called the spliceosome cuts out the introns and joins the exons together.

Splicing also gives the cell a choice. By including or skipping particular exons, alternative splicing lets one gene produce several different mRNAs, and so several different proteins. Most human genes are spliced in more than one way: OpenStax quotes one estimate of about 70%, and later sequencing studies put it at about 95% of genes that have more than one exon. The exons that are kept normally stay in their original order, though; they aren't shuffled.

Reading the code

The mRNA is read in groups of three bases called codons. There are 4 × 4 × 4 = 64 possible codons. Sixty-one of them specify amino acids, and three (UAA, UAG and UGA) are stop codons that end the protein. AUG does double duty: it codes for methionine and also marks where translation starts, so it's called the start codon.

With 61 codons and only 20 amino acids, most amino acids have more than one codon. The code is degenerate (redundant). But it's not ambiguous: each codon means only one thing. UUU and UUC both mean phenylalanine, but UUU never means anything except phenylalanine. The extra codons usually differ only in the third base, which is why many changes to a third base don't change the protein.

The code is also nearly universal: with a few small exceptions, bacteria, plants and people read codons the same way. That's strong evidence that all life shares a common ancestor, and it's why a human gene can be expressed in bacteria.

Translation: tRNA and the ribosome

The adaptor between codon and amino acid is transfer RNA (tRNA). Each tRNA carries a specific amino acid at one end and has a three-base anticodon at the other, which base-pairs with a codon on the mRNA. Enzymes called aminoacyl-tRNA synthetases attach the correct amino acid to each tRNA, using energy from ATP. They're the real translators: if a synthetase loads the wrong amino acid, the ribosome has no way of noticing.

The ribosome is made of a small and a large subunit, each built from rRNA and proteins. The ribosome has three tRNA-binding sites: the A site where a new loaded tRNA arrives, the P site holding the tRNA attached to the growing chain, and the E site where empty tRNAs exit.

1. 1Initiation: the small subunit binds the mRNA and finds the start codon, AUG. An initiator tRNA carrying methionine (a modified form, formyl-methionine, in bacteria) pairs with it. The large subunit then joins, with the initiator tRNA in the P site.
2. 2Elongation: a tRNA whose anticodon matches the next codon enters the A site. The ribosome joins the growing chain to this new amino acid with a peptide bond. That reaction is catalysed by the ribosome's rRNA, not by a protein, so the ribosome is a ribozyme.
3. 3Translocation: the ribosome moves along the mRNA by one codon. The tRNA holding the chain shifts to the P site, the empty tRNA moves to the E site and leaves, and the A site is free for the next tRNA. GTP supplies energy for these steps.
4. 4Termination: when a stop codon reaches the A site, no tRNA matches it. A release factor binds instead, and the finished polypeptide is released. The subunits separate and can be reused.

Many ribosomes can translate the same mRNA at once, one behind another, forming a polyribosome. That's how a single mRNA can churn out many copies of a protein.

Bacteria vs. eukaryotes

Bacteria have no nucleus, so their ribosomes can start translating an mRNA while it's still being transcribed. Transcription and translation are coupled. Bacterial mRNAs don't get caps or poly-A tails, and their genes rarely have introns.

Bacterial mRNAs are often polycistronic: one mRNA carries the code for several proteins, one after another, each with its own start and stop codon. That's how the genes of an operon, such as the lac operon, are switched on together. Eukaryotic mRNAs usually encode just one protein.

• Where: bacteria transcribe and translate in the same compartment; eukaryotes transcribe in the nucleus and translate in the cytoplasm.
• Processing: bacterial mRNA is used as made; eukaryotic pre-mRNA is capped, tailed and spliced.
• RNA polymerases: bacteria have one; eukaryotes have three (I for most rRNA, II for mRNA, III for tRNA and some small RNAs).
• Start: bacteria use formyl-methionine for the first amino acid; eukaryotes use methionine.

Key terms

Transcription / translation

Making RNA from a DNA template / making a polypeptide from mRNA.

Template / coding strand

The DNA strand RNA polymerase reads / the other strand, whose sequence matches the mRNA (T for U).

Promoter

DNA sequence upstream of a gene where RNA polymerase (and its helpers) bind to start transcription.

5′ cap / poly-A tail

Modified G added to the mRNA's 5′ end / run of A's added to its 3′ end; both protect it and help translation.

Intron / exon

Stretch removed from pre-mRNA by splicing / stretch kept in the mature mRNA.

Alternative splicing

Joining different combinations of exons so one gene gives several proteins.

Codon / anticodon

Three-base unit on mRNA / matching three bases on a tRNA.

Degenerate code

Most amino acids have several codons, but each codon has only one meaning.

Polycistronic mRNA

A bacterial mRNA carrying the coding sequences of several proteins.

Check yourself

Try answering in your head before you open each answer.

• 1.A coding strand reads 5′-ATGCCCGGGTAA-3′. Write the mRNA and the template strand, both 5′→3′.Show answerHide

  mRNA: 5′-AUGCCCGGGUAA-3′ (the coding strand with U for T). Template: the complement, written 5′→3′, is 5′-TTACCCGGGCAT-3′. The peptide is Met–Pro–Gly, then stop.

• 2.A scientist puts a human gene, copied straight from the genome, into bacteria. The bacteria make a protein, but it's the wrong length and doesn't work. What's the most likely reason, and how could it be fixed?Show answerHide

  The genomic gene contains introns, and bacteria have no spliceosome, so the introns are translated as if they were code. Using a copy made from the mature mRNA (a cDNA, made with reverse transcriptase), which has no introns, fixes it.

• 3.A mutation removes the 5′ cap-adding step in a eukaryotic cell. Predict two effects on protein production from its mRNAs.Show answerHide

  Uncapped mRNAs would be broken down faster by enzymes, so each mRNA would last a shorter time. And translation would start less efficiently, because the proteins that bring the small ribosomal subunit to the mRNA recognise the cap. Both lower the amount of protein made.