Molecular Biology and Gene Expression

Nutritional Mutants in Neurospora

• George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media.
• Some cells grew when minimal media was supplemented with the amino acid arginine.
• Adrian Srb and Norman Horowitz identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine.
• The results of the experiments provided support for the one gene–one enzyme hypothesis, which states that the function of a gene is to dictate production of a specific enzyme.

The Products of Gene Expression

• Not all proteins are enzymes, so researchers later revised the hypothesis to one gene–one protein.
• Many proteins are composed of several polypeptides, each of which has its own gene.
• Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis.
• It is common to refer to gene products as proteins rather than more precisely as polypeptides.

Basic Principles of Transcription and Translation

• RNA is the bridge between genes and protein synthesis.
• Transcription is the synthesis of RNA using information in DNA.
• Transcription produces messenger RNA (mRNA).
• Translation is the synthesis of a polypeptide, using information in the mRNA.
• Ribosomes are the sites of translation.

Translation

• During translation, the mRNA base triplets, called codons, are read in the 5′ → 3′ direction.
• Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide.
• All 64 codons were deciphered by the mid-1960s.
• Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation.
• The genetic code is redundant (more than one codon may specify a particular amino acid) but not ambiguous (no codon specifies more than one amino acid).
• Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced.

Evolution of the Genetic Code

• The genetic code is nearly universal, shared by the simplest bacteria and the most complex animals.
• Genes can be transcribed and translated after being transplanted from one species to another.
• A language shared by all living things must have been operating very early in the history of life.

Transcription

• Transcription is the first stage of gene expression.
• RNA synthesis is catalyzed by RNA polymerase, which pryes the DNA strands apart and joins together the RNA nucleotides.
• The mRNA is complementary to the DNA template strand.
• RNA polymerase does not need any primer.
• RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine.

Termination of Transcription

• The mechanisms of termination are different in bacteria and eukaryotes.
• In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification.
• In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence.

Eukaryotic Cells Modify RNA After Transcription

• Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm.
• During RNA processing, both ends of the primary transcript are changed.
• Also, in most cases, certain interior sections of the molecule are cut out and the remaining parts spliced together.

Alteration of mRNA Ends

• Each end of a pre-mRNA molecule is modified in a particular way.
• The 5′ end receives a modified nucleotide 5′ cap.
• The 3′ end gets a poly-A tail.
• These modifications share several functions:
  • They seem to facilitate the export of mRNA to the cytoplasm.
  • They protect mRNA from hydrolytic enzymes.
  • They help ribosomes attach to the 5′ end.

Split Genes and RNA Splicing

• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions.
• The noncoding segments in a gene are called intervening sequences, or introns.
• The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences.
• Introns are removed through RNA splicing.
• The removal of introns is accomplished by spliceosomes.
• Spliceosomes consist of a variety of proteins and several small RNAs that recognize the splice sites.
• The RNAs of the spliceosome also catalyze the splicing reaction.

Ribozymes

• Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA.
• Three properties of RNA enable it to function as an enzyme:
  • It can form a three-dimensional structure because of its ability to base-pair with itself.
  • Some bases in RNA contain functional groups that may participate in catalysis.
  • RNA may hydrogen-bond with other nucleic acid molecules.

The Functional and Evolutionary Importance of Introns

• Some introns contain sequences that regulate gene expression and many affect gene products.
• Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing.
• This is called alternative RNA splicing.
• Consequently, the number of different proteins an organism can produce is much greater than its number of genes.
• Proteins often have a modular architecture consisting of discrete regions called domains.
• In many cases, different exons code for the different domains in a protein.
• Exon shuffling may result in the evolution of new proteins by mixing and matching exons between different genes.

Translation: A Closer Look

• Genetic information flows from mRNA to protein through the process of translation.
• A cell translates an mRNA message into protein with the help of transfer RNA (tRNA).
• tRNAs transfer amino acids to the growing polypeptide in a ribosome.
• Translation is a complex process in terms of its biochemistry and mechanics.

The Structure and Function of Transfer RNA

• Each tRNA molecule enables translation of a given mRNA codon into a certain amino acid.
• Each carries a specific amino acid on one end.
• Each has an anticodon on the other end; the anticodon base-pairs with a complementary codon on mRNA.
• A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long.
• Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf.
• Because of hydrogen bonds, tRNA actually twists and folds into a three-dimensional molecule.
• tRNA is roughly L-shaped with the 5′ and 3′ ends both located near one end of the structure.
• The protruding 3′ end acts as an attachment site for an amino acid.

Accurate Translation

• Accurate translation requires two instances of molecular recognition:
  • First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase.
  • Second: a correct match between the tRNA anticodon and an mRNA codon.
• Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to more than one codon.