From Gene to Protein DNA RNA Protein

What Is the Evidence that Genes Code for Proteins? The molecular basis of phenotypes was known before it was known that DNA is the genetic material Studies of many different organisms showed that major phenotypic differences were due to specific proteins

Model organisms - easy to grow or observe; show the phenomenon to be studied Examples: pea plants, Drosophila, E. coli, common bread mold Neurospora crassa

Wild-type strains have enzymes to catalyze all reactions needed to make cell constituents - prototrophs Beadle and Tatum used X-rays as mutagens. Mutants were auxotrophs - needed additional nutrients to grow

For each auxotrophic strain, they found a single compound that would support growth of that strain Suggested the one-gene, one-enzyme hypothesis

Beadle and Tatum found several different arg mutant strains—had to be supplied with arginine arg mutants could have mutations in the same gene; or in different genes that governed steps of a biosynthetic pathway

arg mutants were grown in the presence of compounds suspected to be intermediates in the biosynthetic pathway for arginine This confirmed that each mutant was missing a single enzyme in the pathway

The gene-enzyme relationship has been revised to the one-gene, one-polypeptide relationship Example: In hemoglobin, each polypeptide chain is specified by a separate gene Other genes code for RNA that is not translated to polypeptides; some genes are involved in controlling other genes

Expression of a Gene to Form a Polypeptide Transcription - copies information from gene to a sequence of RNA Translation - converts RNA sequence to amino acid sequence

RNA, ribonucleic acid differs from DNA: Usually one strand The sugar is ribose Contains uracil (U) instead of thymine (T)

RNA can pair with a single strand of DNA, except that adenine pairs with uracil instead of thymine Single-strand RNA can fold into complex shapes by internal base pairing

The central dogma of molecular biology - information flows in one direction when genes are expressed DNA RNA Protein

Messenger RNA (mRNA) forms as a complementary copy of DNA and carries information to the cytoplasm This process is transcription

Within each gene, only one strand of DNA is transcribed - the template strand Transcription produces mRNA; the same process is used to produce tRNA and rRNA

The genetic code - specifies which amino acids will be used to build a protein Codon - a sequence of three bases. Each codon specifies a particular amino acid

Start codon – AUG - initiation signal for translation Stop codons - stops translation and polypeptide is released

For most amino acids, there is more than one codon; the genetic code is redundant each codon specifies only one amino acid

The genetic code is nearly universal - the codons that specify amino acids are the same in all organisms Exceptions: within mitochondria and chloroplasts, and in one group of protists

RNA polymerases catalyze synthesis of RNA RNA polymerases are processive - a single enzyme-template binding results in polymerization of hundreds of RNA bases

Transcription occurs in three phases 1. Initiation 2. Elongation 3. Termination

Initiation Requires a promoter - a special sequence of DNA RNA polymerase binds to the promoter (commonly produces a TATA Box in eukaryotes)

Promoter tells RNA polymerase where to start, which direction to go in, and which strand of DNA to transcribe

Elongation RNA polymerase unwinds DNA about 10 base pairs at a time; reads template in 3′ to 5′ direction The RNA transcript is antiparallel to the DNA template strand RNA polymerases do not proofread and correct mistakes

Termination Specified by a specific DNA base sequence Eukaryotes - first product is a pre-mRNA that is longer than the final mRNA and must undergo processing

Transcription Animation

mRNA modification 1. 5’ cap - modified guanine; protection; recognition site for ribosomes 2. 3’ tail - poly(A) tail (adenine); protection; recognition; transport 3. RNA splicing - exons (expressed sequences) kept introns (intervening sequences) are spliced out forming a spliceosome

RNA Splicing Introns - intervening sequence Non coding Exon - translates to Amino Acids sequence Spliceosome – join together 2 exons that flank the intron Ribozymes – RNA molecules that function as enzymes

RNA Splicing Animation

Evolutionary Importance?? Alternative RNA Splicing Gene gives rise to different proteins depending on which segments are exons during RNA processing Potentially new proteins with new functions Increase chance of crossing over between genes increase genetic recombination

Transfer RNA (tRNA) - an adapter molecule that can bind amino acids, and recognize a nucleotide sequence In the process of translation - tRNA molecules carrying amino acids line up on mRNA in proper sequence for the polypeptide chain

Functions of tRNA: Carries an amino acid Associates with mRNA molecules Interacts with ribosomes

The conformation (3-D shape) of tRNA results from base pairing (H bonds) within the molecule 3′ end is the amino acid attachment site - binds covalently. Always CCA. Anticodon - site of base pairing with mRNA. Unique for each type of tRNA.

Example: DNA codon for arginine: 3′-GCC-5′ Complementary mRNA: 3′-CGG-5′ Anticodon on the tRNA: 3′-GCC-5′ This tRNA is charged with arginine

Ribosome - holds mRNA and tRNA in the correct positions to allow assembly of polypeptide chain Ribosomes are not specific, they can make any type of protein

Ribosomes have two subunits, large and small

How Is RNA Translated into Proteins? Large subunit has three tRNA binding sites: 1. A site binds with anticodon of charged tRNA (carrying an amino acid) 2. P site is where tRNA adds its amino acid to the growing chain 3. E site is where tRNA sits before being released

Hydrogen bonds form between the anticodon of tRNA and the codon of mRNA Small subunit rRNA validates the match - if hydrogen bonds have not formed between all three base pairs, it must be an incorrect match, and the tRNA is rejected

Translation also occurs in three steps 1. Initiation 2. Elongation 3. Termination

Initiation An initiation complex forms - charged tRNA and small ribosomal subunit, both bound to mRNA rRNA binds to recognition site on mRNA “upstream” from the start codon

Start codon is AUG - first amino acid is always methionine (may be removed after translation) The large subunit joins the complex, the charged tRNA is now in the P site of the large subunit

Elongation The second charged tRNA enters the A site Large subunit catalyzes two reactions: 1. Breaks bond between tRNA in P site and its amino acid 2. Peptide bond forms between that amino acid and the amino acid on tRNA in the A site

When the first tRNA has released its methionine, it moves to the E site and dissociates from the ribosome - can then become charged again Elongation occurs as the steps are repeated, assisted by proteins called elongation factors

Termination translation ends when a stop codon enters the A site Stop codon binds a protein release factor - allows hydrolysis of bond between polypeptide chain and tRNA on the P site

Protein Synthesis Animation

Several ribosomes can work together to translate the same mRNA, producing multiple copies of the polypeptide A strand of mRNA with associated ribosomes is called a polyribosome or polysome

What Are Mutations? Somatic mutations occur in somatic (body) cells. Mutation is passed to daughter cells, but not to sexually produced offspring Germ line mutations occur in cells that produce gametes. Can be passed to next generation

All mutations are alterations of the nucleotide sequence Point mutations - change in a single base pair loss, gain, or substitution of a base

Chromosomal mutations - change in segments of DNA loss, duplication, or rearrangement

Point mutations can result from replication and proofreading errors, or from environmental mutagens Silent mutations have no effect on the protein because of the redundancy of the genetic code Silent mutations result in genetic diversity not expressed as phenotype differences

Missense mutations - base substitution results in amino acid substitution

Nonsense mutations - base substitution results in a stop codon

Frame-shift mutations - single bases inserted or deleted usually leads to nonfunctional proteins

Induced mutation - due to an outside agent, a mutagen Chemicals can alter bases (e.g., nitrous acid can cause deamination) Some chemicals add other groups to bases (e.g., benzpyrene adds a group to guanine and prevents base pairing). DNA polymerase will then add any base there

Ionizing radiation such as X-rays create free radicals highly reactive can change bases, break sugar phosphate bonds UV radiation is absorbed by thymine, causing it to form covalent bonds with adjacent nucleotides disrupts DNA replication

Mutation provides the raw material for evolution in the form of genetic diversity Mutations can harm the organism, or be neutral Occasionally, a mutation can improve an organism’s adaptation to its environment, or become favorable as conditions change