BIO 2, Lecture 7 LIFE’S INFORMATION MOLECULE II: TRANSCRIPTION

Overview: The Flow of Genetic Information The information content of DNA is in the form of specific sequences of nucleotides The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Changes in the nucleotide sequence of DNA can lead to changes in the amino acid sequence of proteins The genotype of an organism is comprised of the genes that it carries The phenotype of an organism is comprised of its physical and behavioral traits An organism’s phenotype is dictated, to a large extent, by its genotype Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Genotype: 47(+21) XY Phenotype: Down Syndrome

Genes do not control every aspect of phenotype...

In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway

George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme

EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium

RESULTS Classes of Neurospora crassa Wild type Class I mutantsClass II mutants Class III mutants Minimal medium (MM) (control) MM + ornithine MM + citrulline MM + arginine (control) Condition

CONCLUSION Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Wild type Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C Arginine Gene A Gene B Gene C

Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein But proteins with quarternary structure are encoded by more than one gene, so Beadle and Tatum’s hypothesis was again revised as the one gene–one polypeptide hypothesis

But now we know that each gene can encode more than one polypeptide due to a phenomenon called alternative splicing... So now we have the one gene–one or more polypeptides hypothesis

RNA is the intermediate between genes and the proteins for which they code Transcription is the copying of one strand of the double-stranded DNA into a single- stranded RNA molecule Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide from the mRNA on a ribosome

RNA polymerase Chromosomes are like a library of books (in the form of DNA molecules) that cannot be checked out But an mRNA copy of some of the pages of some of the books (genes) are made in a process called transcription Ribosomes Ribosomes then read the instructions in the RNA molecules to build proteins in a process called translation

In prokaryotes, transcription and translation take place in the same space (the cytosol) and the mRNA produced by transcription is immediately translated without more processing In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

The central dogma is the concept that cells are governed by a cellular chain of command: DNA  RNA  protein (a) Bacterial cell TRANSCRIPTION DNA mRNA TRANSLATION Ribosome Polypeptide

(b) Eukaryotic cell TRANSCRIPTION Nuclear envelope DNA Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide

How are the instructions for assembling amino acids into proteins encoded by the DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA How many bases correspond to an amino acid?

The flow of information from gene to protein is based on a triplet code: a series of non-overlapping, three- nucleotide “words” called codons Example: The triplet 5’-AGT-3’ in a gene results in the placement of the amino acid serine in the polypeptide coded by the gene Another example: 5’-GGG-3’ codes for the animo acid glycine

During transcription, one of the two DNA strands is copied into mRNA During translation, the codons in the mRNA are read in the 5 to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

DNA molecule Gene 1 Gene 2 Gene 3 DNA template strand TRANSCRIPTION TRANSLATION mRNA Protein Codon Amino acid DNA coding strand

All 64 codons were deciphered by the mid-1960s (4 3 = 64) Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation of the mRNA The genetic code is redundant but not ambiguous More than one codon can code for one amino acid No codon specifies more than one amino acid

Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon)

The genetic code is universal, shared by the simplest bacteria to the most complex animals This is why genes can be transcribed and translated after being transplanted from one species to another (recombinant DNA technology)

Pig expressing a jellyfish gene

Transcription, the first stage of gene expression, has been examined in great detail RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine

The DNA sequence where RNA polymerase attaches to a gene is called a promoter because the presence of this sequence “promotes” the recognition and transcription of the gene Areas of the DNA lacking promoters are not transcribed The stretch of DNA that is transcribed is called a transcription unit

Promoter Transcription unit DNA Start point RNA polymerase

Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA

Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA 2 Elongation Rewound DNA RNA transcript

Promoter Transcription unit DNA Start point RNA polymerase Initiation RNA transcript 5 5 Unwound DNA Template strand of DNA 2 Elongation Rewound DNA RNA transcript 3 Termination Completed RNA transcript

Elongation RNA polymerase Nontemplate strand of DNA RNA nucleotides 3 end Direction of transcription (“downstream”) Template strand of DNA Newly made RNA 3 5 5

Transcription can be broken down into 3 stages: –Initiation –Elongation –Termination

Promoters attract proteins called transcription factors to the gene Transcription factors then attract RNA polymerase so that transcription can be initiated The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex Promoters contain A-T rich regions, making it easier for RNA polymerase to pry apart (“melt”) the DNA strands

A eukaryotic promoter includes a TATA box Promoter TATA box Start point Template DNA strand Transcription factors Several transcription factors must bind to the DNA before RNA polymerase II can do so Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. RNA polymerase II Transcription factors RNA transcript Transcription initiation complex

As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases

The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the growing RNA chain; the polymerase eventually falls off the DNA

Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm During RNA processing, both ends of the primary transcript are usually altered Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

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 –They protect mRNA from hydrolytic enzymes –They help ribosomes attach to the 5 end

Protein-coding segment Polyadenylation signal 3 3’ UTR5’ UTR 5 5’ Cap Start codon Stop codon Poly-A tail G PPPAAUAAA AAA … Structure of a eukaryotic mRNA

Most eukaryotic genes have long noncoding stretches of nucleotides that lie between coding regions These noncoding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

Watson and Crick reasoned that the pairing was more specific, dictated by the base structures They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C

Pre-mRNA mRNA Coding segment Introns cut out and exons spliced together 5’ Cap Exon Intron 5’ ExonIntron 105 Exon 146 3’ Poly-A tail 5’ Cap 5’ UTR3’ UTR 1 146

Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Such variations are called alternative RNA splicing Because of alternative splicing, 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

Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

Gene DNA Exon 1Exon 2 Exon 3 Intron Transcription RNA processing Translation Domain 2 Domain 3 Domain 1 Polypeptide