8.4 Protein Synthesis

8.4 Protein Synthesis

A cell builds a polypeptide in two transfers, with RNA processing between them in eukaryotes. Transcription copies a gene from DNA into RNA. Translation reads that RNA and assembles amino acids. Which genes are transcribed, how the transcript is finished, and whether ribosomes initiate are the controls of protein synthesis.

Transcription Copies DNA into RNA

RNA polymerase builds the RNA. It is recruited at a promoter, the DNA region where transcription of that gene can start. In bacteria, sigma factor helps the polymerase recognize promoter sequences upstream of the start site. In eukaryotes, transcription factors assemble at the promoter, and RNA polymerase II makes messenger RNA. If RNA polymerase is not recruited, that gene yields no new transcript, so the promoter is a control point for which proteins the cell makes.

The polymerase reads the template strand from 3-prime toward 5-prime, and the RNA grows 5-prime to 3-prime. Unlike DNA polymerase, RNA polymerase needs no primer. RNA nucleotides contain ribose, and they use uracil where DNA would use thymine. The other DNA strand is the coding strand. It matches the RNA, with thymine wherever the RNA has uracil. The polymerase does not read the coding strand. Treating the coding strand as the template reverses the two names.

Where the template is 3'-TAC-5', the RNA is 5'-AUG-3', and the coding strand is 5'-ATG-3'. Swap that thymine for uracil and the coding strand spells the RNA. Transcription has copied DNA into RNA. It has not built a polypeptide.

Eukaryotic Processing of the Primary Transcript

The eukaryotic primary transcript is often much longer than the mature mRNA. Three processing events finish it before it leaves the nucleus.

A 5-prime cap, 7-methylguanosine, is added to the 5-prime end. It helps the ribosome recognize the message and protects that end from degradation. At the 3-prime end the transcript is cleaved, and poly-A polymerase adds a poly-A tail, a run of adenine nucleotides. The tail is not copied from a long stretch of thymine in the gene. The signal AAUAAA commonly marks where cleavage and tail addition occur. Because the tail influences how long the mRNA lasts, cap and tail also affect how much protein can be made.

Spliceosomes remove introns. The exons remain and are joined. The intron is typically released as a lariat and then degraded. The cuts must be exact, because an error of one nucleotide would shift every later codon. Alternative splicing keeps different exon combinations from one primary transcript, so one gene can yield more than one polypeptide. That choice changes the protein without changing the DNA sequence. It is post-transcriptional control.

Prokaryotes generally do not process mRNA this way. With no nucleus between the polymerase and the ribosomes, translation of an mRNA can begin before transcription of that mRNA finishes. The two processes are coupled. Bacterial mRNA is not given a 7-methylguanosine cap and is not spliced by spliceosomes. It is often polycistronic, with several coding sequences on one message. A typical eukaryotic mRNA is monocistronic.

The Genetic Code

The ribosome reads codons, non-overlapping groups of three bases. Four bases in groups of three give 64 codons. The code is redundant: most of the 20 amino acids have more than one codon, often differing at the third base, which is why many silent mutations are third-base changes. The assignments are nearly universal in animals, plants, fungi, and bacteria, with exceptions in some mitochondrial genomes.

AUG is the start codon and codes for methionine. Bacteria usually begin with N-formylmethionine, but the mRNA codon is still AUG. UAA, UAG, and UGA are stops. They do not code for an amino acid. The other 61 codons specify the twenty amino acids. Counting threes from AUG sets the reading frame.

Translation Builds the Polypeptide

Translation uses the mRNA, the ribosome, and tRNA. The ribosome is ribosomal RNA plus proteins, and its peptidyl transferase activity is a ribozyme: ribosomal RNA forms the peptide bond. Each tRNA carries an amino acid and an anticodon that pairs with the codon. Pairing is antiparallel, so the codon 5'-AUG-3' matches the anticodon 3'-UAC-5'. The anticodon is on the tRNA, not on the mRNA.

Aminoacyl-tRNA synthetases charge each tRNA before it reaches the ribosome. A synthetase attaches one amino acid to the 3-prime end of the matching tRNA, using ATP. At the ribosome the check is the anticodon-codon pair. The ribosome does not recheck the amino acid, so a mischarged tRNA is accepted whenever its anticodon matches.

Initiation places AUG in the P site. In eukaryotes the small subunit and the initiator tRNA, carrying methionine, bind near the cap and scan, usually to the first AUG, before the large subunit joins. In bacteria the Shine-Dalgarno sequence upstream of AUG base-pairs with small-subunit ribosomal RNA and positions the start codon. The bacterial initiator tRNA carries N-formylmethionine and also sits in the P site. Later charged tRNAs do not enter at the P site.

During elongation the A site receives the next charged tRNA, the one whose anticodon matches the codon shown there. The chain on the P-site tRNA is transferred onto that new amino acid. The ribosome then moves three nucleotides toward the 3-prime end of the mRNA. The tRNA holding the chain now occupies the P site, and the empty tRNA leaves from the E site. The polypeptide grows from its amino end toward its carboxyl end.

A stop codon has no tRNA and codes for no amino acid. A release factor binds UAA, UAG, or UGA in the A site. The finished polypeptide is released from the tRNA in the P site, and the subunits separate. No amino acid is inserted at a stop.

Those steps are also the controls. Transcription starts only when RNA polymerase is recruited to the promoter. In eukaryotes, splicing chooses which exons remain, and the cap and tail affect how long the mRNA can be translated. Initiation decides whether a ribosome begins at AUG.