Chapter 13: from Gene to Protein

Chapter 13: From gene to protein

Outline
·  Metabolic defects provided evidence that genes specify proteins
·  Transcription and translation are the two main steps from gene to protein
·  The genetic code
·  Transcription
o  initiation
o  elongation
o  termination
·  Translation is RNA-directed synthesis of a polypeptide
o  Ribosomes
o  Translation divided into 3 parts
§  initiation
§  elongation
§  termination
·  Mutations
o  substitutions
o  deletions
o  insertions

DNA inherited by an organism leads to specific traits by dictating the synthesis of certain proteins. Proteins are the link between genotype and phenotype. This chapter explores the steps in the flow of information from genes to proteins.

Metabolic defects provided evidence that genes specify proteins

·  Archibald Garrod (1909): inferred from "inborn diseases of metabolism" that genes code for enzymes that catalyze specific reactions in cells.

o  E.g. hereditary alkaptonuria (urine turns black upon exposure to air) caused by lack of enzyme that metabolizes alkapton.

·  Beadle and Tatum (late 1930's): used auxotrophic mutants of Neurospora crassa ( a type of fungus) to prove Garrods hypothesis that genes encode enzymes (i.e. one-gene-one-enzyme hypothesis).

o  Wildtype N. crassa normally grows well in minimal medium (salts, glucose, and biotin). Beadle and Tatum identitified mutants which could not grow in minimal medium. These mutants grew only if supplied with all amino acids. N. crassa is a model organism. It is haploid, so its easy to select for recessive mutants. In diploids, normal allele masks mutant recessive alleles, so its difficult to select based on phenotype.

o  Auxotroph = nutritional mutant which can not grow on minimal medium. Grow only if supplied with missing nutrient. (e.g. amino acid which they can not synthesize on their own).

o  Refer to Fig 17.2: Beadle and Tatum mutated N. crassa and then looked for mutants that differed in their nutritional needs from the wildtype. They used replica-plating to pinpoint auxotrophic defects by determining precisely which nutrients had to be added to media to allow growth. They chose to study Arginine mutants.

§  Using complementation tests, identified three different classes of mutants which lacked ability to synthesize the amino acid arginine. They suspected that each class of mutants lacked a different enzyme in the metabolic pathway from some precursor to arginine.

§  By plating each class of mutant on minimal media containing a different intermediate in the metabolic pathway leading to arginine, they showed that the different classes of mutants were blocked at a different enzymatic step in the pathway. It was subsequently shown that each mutant in fact lacked a particular enzyme in the arg pathway.

§  These experiments also showed that the combination of genetics with biochemistry could be used to work out the steps in metabollic pathways.

·  One-gene-one polypeptide hypothesis: further experimentation showed that genes encode for proteins, not all of which are enzymes. Also, many proteins have a quaternary structure, and each subunit is encoded by a different gene.

Transcription and translation are the two main steps from gene to protein

·  Soon after the discovery of the double helix, Francis Crick proposed that biological information flows from DNA to RNA to protein. This became known as the central dogma of biology. It was later modified to incorporate the fact that in some viruses, RNA is copied into DNA by reverse transcriptases.

·  Genes are instructions for making specific proteins. But proteins are not made from DNA directly. The flow of information from DNA to protein proceeds through a RNA intermediate. DNA is first transcribed (copied) into mRNA which is then translated into protein.

·  RNA differs structurally from DNA in 3 ways:

o  1. Has uracyl instead thymine

o  2. Sugar is a ribose not a deoxyribose (RNA has OH at 2' C)

o  3. RNA is single stranded (although it can form Watson-Crick base-pairing).

·  Nucleic acids are linear molecules made from 4 monomers (nucleotides). Proteins are also polymers, but made from 20 different amino acids.

·  How does DNA code for protein? Two major steps:

o  1. Transcription: synthesis of complementary RNA from coding strand of DNA.

§  this RNA molecule is called a messenger RNA (mRNA)

§  mRNA is synthesized by RNA polymerase.

§  mRNA and DNA have same language (i.e. code).

o  2. Translation: synthesis of polypeptide under direction of mRNA.

§  involves a change from DNA language into protein language.

§  involves mRNA, ribosomes, transfer RNA (tRNA)

·  The basic mechanisms of transcription and translation are the same for both eukaryotes and prokaryotes. However, there are some differences (Fig 17.3)

o  1. Prokaryotes lack a nucleus; transcription and translation are coupled, i.e. ribosomes translate mRNA while transcription is still in progress. In eukaryotes, mRNA must exit the nucleus to be translated in cytoplasm.

o  2. Differences in gene structure means that transcripts must be processed in eukaryotes, but not in prokaryotes.

o  3. In eukaryotes, each gene is transcribed separately. In Prokaryotes, many genes can be transcribed into a single mRNA (polycistronic RNA).

The genetic code

·  DNA code is a triplet code, i.e. each 3 consecutive bases codes for one amino acid (Fig 17.4).

·  64 possible "words" can be made from a triplet code (4x4x4=64). Since there are 64 possible code words, and only 20 amino acids, some amino acids are encoded by more than one code word. Thus the genetic code is redundant (but not ambiguous).

·  The genetic instructions for a polypeptide chain are written in the DNA as a series of three-nucleotide words.

·  For each gene, only one of the two strands is transcribed. This is called the template or noncoding strand. The other strand functions to make complementary strand during DNA replication.

·  mRNA is complementary to DNA template. The mRNA base triplets are called codons.

·  During translation, the sequence of codons along a genetic message (mRNA) is decoded, or translated, into a sequence of amino acids making the polypeptide chain. Each codon along the mRNA molecule specifies which of the 20 amino acids will be incorporated at the corresponding position along a polypeptide. It takes 900 nucleotides along a mRNA strand to make a polypeptide 300 amino acids long.

Codon Table (Fig 17.5)

·  Not all codons code for amino acids
o  AUG codes for methionine but also serves as start codon, i.e. signals start site for translation.
o  three codons, (UAA, UAG, UGA) are stop codons, i.e. signal the end of translation.
·  Ability to extract intended message from written language depends on reading the symbols in the correct sequence of groupings. This ordering is called the reading frame.
o  change in reading frame results in synthesis of different polypeptide.
o  RNA polymerase uses start codon to establish proper reading frame. All polypeptides have methionine as first amino acid.
·  All organisms have the same genetic code, i.e. genetic code is universal
o  most compelling piece of evidence suggesting that all living things have evolved from a common ancestor.
o  Also means that genes from humans can be cloned into other species and they will specify the same polypeptide. Useful in biotechnology and genetic engineering.

Transcription

·  Coding stand of DNA is transcribed into mRNA by the enzyme RNA polymerase (Fig 17.7 and Fig 17.8).

·  RNA polymerase: binds promoter region of genes, pries apart DNA strands, and joins together RNA nucleotides as they base pair along DNA template. Adds nucleotides only in the 5' to 3' direction. Stops transcription when it reaches sequences at end of gene.

·  Transcription occurs in 3 stages:

o  1. RNA binding and initiation of transcription:

§  RNA polymerase binds promoter region of gene, recognizes speficic DNA sequences, pries DNA strands apart and begins polymerization

§  Promoter = DNA sequences upstream of genes responsible for binding RNA polymerase and transcription factors.

§  transcription factors = proteins which bind promoters and RNA polymerase. Determine when, where, and how much mRNA is produced.

o  2. Elongation of RNA strand:

§  RNA polymerase moves along DNA, untwisting helix, separating the DNA strands (10 bases at a time) and joins RNA nucleotides together as they base pair to template strand.

§  as mRNA is made, it peels away from template

§  rate of transcription is about 60 bases/sec.

o  3. Termination of transcription:

§  RNA polymerase reaches termination sequence at end of gene and stops polymerizing.

RNA processing in eukaryotes

·  Eukaryotic cells modify their primary transcripts in the nucleus before the transcripts are dispatched into the cytoplasm to be translated into proteins. These modifications are referred to as RNA processing (Fig 17.9). The pre-mRNA undergoes 3 modifications:

·  Addition of a 5' CAP
o  a modified guanine nucleotide is added to 5' end of transcript.
o  5' cap promotes export into cytoplasm, protects mRNA from degredation, and is necessary for translation.
·  Addition of a poly-A tail
o  An enzyme adds 50-250 adenine nucleotides to 3' end of transcript, forming poly-A tail.
o  Poly-A tail functions in transport of transcript out of nucleus, and protects mRNA from degredation.
o  Poly-A tail is not translated into polypeptide.
·  Removal of introns

o  Eukaryotic genes contain noncoding sequences that are not translated, called introns. The coding sequences that are translated are called exons (Fig 17.10). The number of introns and exons varies between different genes.
o  The removal of introns from primary transcripts is called RNA splicing, and is carried out by a group of ribonucleoproteins which assemble to form the spliceosome. Spliceosomes recognize the intron/exon border sequences, remove the intervening intron, and then join the exons together, to form a contiguous coding sequence that is ready for translation (Fig 17.10 - 11).
o  As a result of RNA splicing, primary transcripts are usually much longer than mature transcripts.
o  Alternative splicing can lead to different proteins being encoded by the same gene. Because of alternative splicing, the number of different proteins made by an organism is much larger than the number of genes it encodes.

Translation is RNA-directed synthesis of a polypeptide

·  In translation, message in mRNA gets translated (interpreted) into protein. The interpreter is another type of RNA molecule called transfer RNA (tRNA).

o  tRNA = functions to transfer amino acids from the cytoplasm's amino acid pool to a ribosome. The ribosome adds each amino acid brought to it by tRNA to the growing end of a polypeptide chain (Fig 17.13)

Structure and function of tRNA (Fig 17.14

o  transcribed from DNA
o  consists of single RNA strand (~ 80 bases long)
o  folds back onto itself to form secondary structure (cloverleaf-like)
o  3' end of tRNA binds to specific amino acid. Each amino acid binds to a different tRNA. Each type of tRNA associates with a particular mRNA codon. This association is based on H-bonding between codon and complementary bases in anticodon of tRNA.
§  A tRNA that binds to an mRNA codon by specifying a particular amino acid must carry only that amino acid to the ribosome.
§  Each amino acid is matched with the correct tRNA by a specific enzyme called aminoacyl-tRNA synthase (Fig 17.15). There's a whole family of these enzymes, one for each amino acid.

· 

Ribosomes

·  Ribosome holds the tRNA and mRNA molecules close together and catalyzes the addition of an amino acid to the carboxyl end of a growing polypeptide(Fig 17.16).

·  Made of two subunits (large and small). In eukaryotes, ribosomes are made in nucleolus.
·  Each subunit is an aggregate of numerous proteins associated with another type of RNA called ribosomal RNA (rRNA).
·  Has binding site for mRNA and two binding sites for tRNA, known as P site (peptidyl-tRNA site) and A site (aminoacyl-tRNA site)

Translation divided into 3 parts:

·  1. Intitiation (Fig 16.17)

o  Small subunit of ribosome binds mRNA and then binds initiator tRNA at the first codon. This establisheds the reading frame.

o  Large subunit then binds, and exposes the next codon on mRNA at the A site of ribosome. Ready for elongation.

·  2. Elongation (Fig 16.18)

o  1. codon recognition: an incoming aminoacyl-tRNA binds to codon at A-site

o  2. peptide bond formation: peptide bond is formed between new amino acid and growing polypeptide chain.

o  3. Translocation; tRNA that was in P site is released. tRNA in the A site is translocated to the P site. In the process, ribosome advances by one codon.

·  3 Terminatiuon (Fig 17.19):

o  Stop codon reached along mRNA.; ribosome binds a protein called release factor which hydrolyzes bond between tRNA at P site and last amino acid of polypeptide. Both tRNA and polypeptide float away.

·  During and after its synthesis, polypeptide chain begins to coil and fold spontaneously, to form functional proteins with a specific conformation.

·  Some polypeptides have signal sequences that target them to specific destinations in the cell (Fig 17.21)

Mutations

·  Mutations are changes inthe genetic makeup of a cell.

·  Ultimate source of all genetic variation.

·  Point mutations = changes in just one nucleotide in a single gene. Three types: substitutions, insertions, and deletions

o  Base Pair substitutions (Fig 17.24).

o  replacement of one nucleotide by another; may or may not affect amino acid sequence (depends on specific substitution)
o  silent mutation: new codon still codes for same amino acid (aka synonomous substitution)(b/c code is degenerate).
o  missence mutation; codon changed such that it encodes a new amino acid ( aka nonsynonomous substitution) .
o  nonsense mutation: codon changed to stop codon; causes early chain termination.

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