BIO 2, Lecture 7BIO 2, Lecture 7BIO 2, Lecture 7BIO 2, Lecture 7LIFE’S INFORMATION LIFE’S INFORMATION

MOLECULE II: TRANSCRIPTIONMOLECULE 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

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• 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) XYPhenotype: 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-typecells growingand dividing

No growth:Mutant cellscannot growand divide

Minimal medium

RESULTSClasses of Neurospora crassa

Wild type Class I mutantsClass II mutantsClass III mutants

Minimalmedium(MM)(control)

MM +ornithine

MM +citrulline

MM +arginine(control)

Con

dit

ion

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 Precursor Precursor PrecursorEnzyme AEnzyme AEnzyme AEnzyme A

Ornithine Ornithine Ornithine OrnithineEnzyme BEnzyme B Enzyme BEnzyme B

Citrulline Citrulline Citrulline CitrullineEnzyme CEnzyme CEnzyme CEnzyme C

Arginine Arginine Arginine 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

TRANSLATIONRibosome

Polypeptide

(b) Eukaryotic cell

TRANSCRIPTION

Nuclearenvelope

DNA

Pre-mRNARNA 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

DNAmolecule

Gene 1

Gene 2

Gene 3

DNAtemplatestrand

TRANSCRIPTION

TRANSLATION

mRNA

Protein

Codon

Amino acid

DNAcodingstrand

• All 64 codons were deciphered by the mid-1960s (43 = 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

Fir

st

mR

NA

base (

5 e

nd

of

cod

on

)

Th

ird

mR

NA

base (

3 e

nd

of

cod

on

)

• 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

DNAStart point

RNA polymerase

553

3

Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

5 5

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

5 5 3 3 3

RNAtranscript

3 Termination

5

5

5 33

3Completed RNA transcript

Elongation

RNApolymerase

Nontemplatestrand of DNA

RNA nucleotides

3 end

Direction oftranscription(“downstream”) Template

strand of DNA

Newly madeRNA

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 promoterincludes a TATA box

3

1

2

3

Promoter

TATA box Start point

Template

TemplateDNA strand

535

Transcriptionfactors

Several transcription factors mustbind to the DNA before RNApolymerase II can do so.

5533

Additional transcription factors bind tothe DNA along with RNA polymerase II,forming the transcription initiation complex.

RNA polymerase IITranscription factors

55 53

3

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 segmentPolyadenylation signal3

3’ UTR5’ UTR

5

5’ Cap Start codon Stop codon Poly-A tail

G P PP AAUAAA AAA 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

Codingsegment

Introns cut out andexons spliced together

5’ Cap

Exon Intron5’

1 30 31 104

Exon Intron

105

Exon

146

3’Poly-A tail

Poly-A tail5’ Cap

5’ UTR 3’ UTR1 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

GeneDNA

Exon 1 Exon 2 Exon 3Intron Intron

Transcription

RNA processing

Translation

Domain 2

Domain 3

Domain 1

Polypeptide