Page 117 - Genetics_From_Genes_to_Genomes_6th_FULL_Part2

276 Chapter 8 Gene Expression: The Flow of Information from DNA to RNA to Protein

messenger RNA and the development of techniques for Figure 8.5 How geneticists used synthetic mRNAs to
synthesizing simple messenger RNA molecules had to limit the coding possibilities. (a) Poly-U mRNA generates a
occur first so that researchers could manufacture simple poly-phenylalanine polypeptide. (b) Polydi-, polytri-, and polytetra-
proteins in the test tube. nucleotides encode simple polypeptides. Some synthetic mRNAs,
such as poly-GUAA, contain stop codons in all three reading frames
and thus specify the construction only of short peptides.
The discovery of messenger RNAs (a) Poly-U mRNA encodes polyphenylalanine.
In the 1950s, researchers exposed eukaryotic cells to amino
acids tagged with radioactivity and observed that protein
synthesis incorporating the radioactive amino acids into 5' UUUUUUUU N ... Phe Phe Phe Phe Phe Phe Phe ... C
polypeptides takes place in the cytoplasm, even though the Analyze radioactive
genes for those polypeptides are sequestered in the cell nu­ polypeptides synthesized
cleus. From this discovery, they deduced the existence of an Synthetic 3' In vitro translational
intermediate molecule, made in the nucleus and capable of mRNA system plus radioactive
transporting DNA sequence information to the cytoplasm, amino acids
where it can direct protein synthesis. RNA was a prime can­ (b) Analyzing the coding possibilities.
didate for this intermediary information­carrying molecule.
Because of RNA’s potential for base pairing with a Synthetic mRNA Polypeptides synthesized
strand of DNA, one could imagine the cellular machinery Polypeptides with one amino acid
copying a strand of DNA into a complementary strand of poly-U UUUU … Phe-Phe-Phe …
RNA in a manner analogous to the DNA­to­DNA copying poly-C CCCC … Pro-Pro-Pro …
Lys-Lys-Lys …
poly-A AAAA …
of DNA replication. Subsequent studies in eukaryotes us­ poly-G GGGG … Gly-Gly-Gly …
ing radioactive uracil, a base found only in RNA, showed
that although the molecules are synthesized in the nucleus, Repeating dinucleotides Polypeptides with alternating amino acids

at least some of them migrate to the cytoplasm. Among poly-UC UCUCUC … Ser-Leu-Ser-Leu …
those RNA molecules that migrate to the cytoplasm are the poly-AG AGAGAG … Arg-Glu-Arg-Glu …
poly-UG UGUGUG …
Cys-Val-Cys-Val …
messenger RNAs, or mRNAs, depicted in Fig. 8.1. They poly-AC ACACAC … Thr-His-Thr-His …
arise in the nucleus from the transcription of DNA se­ Repeating trinucleotides Three polypeptides each with one amino
quence information and then move (after processing) to the acid
cytoplasm, where they determine the order of amino acids
during protein synthesis. poly-UUC UUCUUCUUC … Phe-Phe.... and Ser-Ser.... and Leu-Leu....
Lys-Lys.... and Arg-Arg.... and Glu-Glu....
poly-AAG AAGAAGAAG …
poly-UUG UUGUUGUUG … Leu-Leu.... and Cys-Cys.... and Val-Val....
poly-UAC UACUACUAC … Tyr-Tyr.... and Thr-Thr.... and Leu-Leu....
Using synthetic mRNAs and in vitro translation Repeating tetranucleotides Polypeptides with repeating units of four
Knowledge of mRNA served as the framework for two ex­ amino acids
perimental breakthroughs that led to the deciphering of the poly-UAUC UAUCUAUC … Tyr-Leu-Ser-Ile-Tyr-Leu-Ser-Ile...
genetic code. In the first, biochemists obtained cellular ex­ poly-UUAC UUACUUAC … Leu-Leu-Thr-Tyr-Leu-Leu-Thr-Tyr...
tracts that, with the addition of mRNA, synthesized poly­ poly-GUAA GUAAGUAA … none
poly-GAUA GAUAGAUA …
none
peptides in a test tube. They called these extracts in vitro
translational systems. The second breakthrough was the
development of techniques enabling the synthesis of artifi­
cial mRNAs containing only a few codons of known com­ The chemist Har Gobind Khorana later made mRNAs
position. When added to in vitro translational systems, with repeating dinucleotides, such as poly­UC (5′. . .
these simple, synthetic mRNAs directed the formation of UCUCUCUC . . . 3′), repeating trinucleotides, such as poly­
simple polypeptides. UUC, and repeating tetranucleotides, such as poly­UAUC,
In 1961, Marshall Nirenberg and Heinrich Matthaei and used them to direct the synthesis of slightly more com­
added a synthetic poly­U (5′. . . UUUUUUUUUUUU . . . 3′) plex polypeptides. As Fig. 8.5b shows, his results limited
mRNA to a cell­free translational system derived from the coding possibilities, but some ambiguities remained.
E. coli. With the poly­U mRNA, phenylalanine (Phe) was For example, poly­UC encodes the polypeptide N . . . Ser­
the only amino acid incorporated into the resulting poly­ Leu­Ser­Leu­Ser­Leu . . . C in which serine and leucine al­
peptides (Fig. 8.5a). Because UUU is the only possible ternate with each other. Although the mRNA contains only
triplet in poly­U, UUU must be a codon for phenylalanine. two different codons (5′ UCU 3′ and 5′ CUC 3′), it is not
In a similar fashion, Nirenberg and Matthaei showed that obvious which corresponds to serine and which to leucine.
CCC encodes proline (Pro), AAA is a codon for lysine Nirenberg and Philip Leder resolved these ambiguities
(Lys), and GGG encodes glycine (Gly) (Fig. 8.5b). in 1965 with experiments in which they added short,