Transcription & Translation

From Gene to Protein

Part 1

A little history lesson

• 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

Nutritional Mutants in Neurospora: Scientific

Inquiry

• 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

• However, some proteins aren’t enzymes, so researchers later revised the hypothesis: 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

• Note that it is common to refer to gene products as proteins rather than polypeptides

Part 2

A Basic Overview

Background

• The process that describes how enzymes and other proteins are made from DNA is called protein synthesis.

• Protein Synthesis has 3 steps:– Transcription

• mRNA is created from a strand of DNA

– RNA processing• mRNA is edited

– Translation• mRNA is read by a ribosome and used to assemble amino

acids into polypeptides

Types of RNA

• mRNA– Messenger RNA

– A single strand of RNA that provides a template used for sequencing amino acids into a polypeptide.

– A triplet of 3 adjacent nucleotides on the mRNA, called a codon, codes for one specific amino acid.

– Since there are 64 possible ways that 4 nucleotides can be arranged in triplet combinations, there are 64 possible codons.

– However, there are only 20 amino acids. Thus, some codons code for the same amino acid.

Genetic Code

• A visual representation of the possible

codon combinations and the amino acids

each codon codes for.

Fig. 17-5

Second mRNA base

Fir

st

mR

NA

ba

se

(5e

nd

of

co

do

n)

Th

ird

mR

NA

ba

se

(3e

nd

of

co

do

n)

Genetic Code

Pinwheel Genetic Code

Types of RNA

• tRNA– Transfer RNA

– A short RNA molecule used for transporting amino acids to their proper place on the mRNA template.

– There are about 45 different tRNAs

– Due to interactions between various parts of the tRNA molecule, it is folded and looks like the 3 leaflets of a clover leaf.

– Contains the anticodon that allows it to bind to mRNA.

• Example: if mRNA has the codon ‘AUG’, tRNA will have the complimentary anticodon ‘UAC’. This codon-anticodon arrangement allows the tRNA to connect to the mRNA.

Types of RNA

• rRNA

– ribosomal RNA

– The building blocks of ribosomes

– In the nucleolus, various proteins are imported from the cytoplasm and assembled with rRNA to form large and small ribosomal subunits.

– Together these two subunits form a ribosome that coordinates the activities of mRNA and tRNA during translation.

Transcription

3 Parts

• 1.) Initiation• RNA polymerase attaches to a promoter region on

the DNA and begins to unzip the two strands.

• The promotor region for mRNA transcriptions often

contains the sequence T-A-T-A. called the TATA

Box.

• 2.) Elongation• Occurs as the RNA pol unzips the DNA and

assembles RNA nucleotides using one side of the

DNA as a template.

• Elongation occurs in the 5’3’ direction

• 3.) Termination• Occurs when the RNA pol reaches a special

sequence of nucleotides that serve as a

termination point.

• In eukaryotes, the termination region often

contains the DNA sequence AAAAAAA.

Fig. 17-7a-1Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Fig. 17-7a-2Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

55

UnwoundDNA

Template strandof DNA

Fig. 17-7a-3Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

55

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

553 3 3

RNAtranscript

Fig. 17-7a-4Promoter Transcription unit

DNAStart point

RNA polymerase

553

3

Initiation

33

1

RNAtranscript

55

UnwoundDNA

Template strandof DNA

2 Elongation

RewoundDNA

5

553 3 3

RNAtranscript

3 Termination

5

5

533

3Completed RNA transcript

Fig. 17-7b

Elongation

RNA

polymerase

Nontemplatestrand of DNA

RNA nucleotides

3 end

Direction oftranscription(“downstream”) Template

strand of DNA

Newly madeRNA

3

5

5

Animation

• Transcription Animation

mRNA Processing

Editing the message

Steps of processing

• 1.) The 5’ cap (-P-P-P-G-5’)_• A cap is added to the 5’ end of the mRNA.

• The cap is a guanine nucleotide with 2 additional phosphate groups, forming GTP (similar to ATP).

• This capping gives stability to the mRNA and an attachment point for the ribosome.

• 2.) The poly-A tail (-A-A-A….A-A-3’)• A poly-A tail is added to the 3’ end of the mRNA.

• The tail consists of about 200 adenine nucleotides.

• Provides stability and aids in the mRNA passing through the nuclear envelope.

• 3.) RNA Splicing• Nucleotide segments are removed from the mRNA

• DNA segments contain both coding and non-coding sequences.

• The coding segments = exons;

• The non-coding segments = introns

• The original unprocessed mRNA contains both the coding and non-coding sequences.

• The introns have to be cut out and the exons have to be spliced together in order to create an mRNA with a continuous coding sequence.

Fig. 17-10

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

• In some cases, RNA splicing is carried

out by spliceosomes

• Spliceosomes consist of a variety of

proteins and several small nuclear

ribonucleoproteins (snRNPs) that

recognize the splice sites

Fig. 17-11-1

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

Protein

snRNA

snRNPs

Otherproteins

5

Fig. 17-11-2

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

Protein

snRNA

snRNPs

Otherproteins

5

5

Spliceosome

Fig. 17-11-3

RNA transcript (pre-mRNA)

Exon 1 Exon 2Intron

Protein

snRNA

snRNPs

Otherproteins

5

5

Spliceosome

Spliceosomecomponents

Cut-outintron

mRNA

Exon 1 Exon 25

Animation

• RNA Splicing Animation

• Splicing Animation #2

• Another RNA Splicing Animation

Ribozymes

• Ribozymes are catalytic RNA molecules

that function as enzymes and can splice

RNA

• The discovery of ribozymes rendered

obsolete the belief that all biological

catalysts were proteins

• 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

– RNA may hydrogen-bond with other nucleic

acid molecules

The Functional and Evolutionary

Importance of Introns• 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

Translation

Making a protein

Background

• After transcription, the mRNA, tRNA and

ribosomal subunits are transported across

the nuclear envelope and into the

cytoplasm.

• In the cytoplasm, the amino acids attach to

the 3’ end of the tRNAs, forming an

aminoacyl-tRNA.

Background

• The reaction to attach the amino acid to

the tRNA requires an enzyme specific to

each tRNA and the energy from one ATP.

• As in transcription, translation is categorized into

3 steps:

– Initiation

– Elongation

– Termination

• The energy for translation is provided by several

GTP molecules.

– GTP acts as an energy supplier in the same manner

as ATP

• A ribosome has three binding sites for

tRNA:

– The P site holds the tRNA that carries the

growing polypeptide chain

– The A site holds the tRNA that carries the next

amino acid to be added to the chain

– The E site is the exit site, where discharged

tRNAs leave the ribosome

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Steps of translation

• 1.) Initiation begins when the small ribosomal subunit attaches to a special region near the 5’ end of the mRNA.

• 2.) A tRNA (with the anticodon UAC) carrying the amino acid methionine attaches to the mRNA at the start codon, AUG, on a spot on the ribosome called the “P” site

Fig. 17-17

3

35

5U

U

AA

C

G

GTP GDPInitiator

tRNA

mRNA

53

Start codon

mRNA binding site

Smallribosomalsubunit

5

P site

Translation initiation complex

3

E A

Largeribosomalsubunit

• 3.) Another tRNA carrying another amino

acid comes in and binds to the mRNA

at the “A”site.

• 4.) The amino acid from the tRNA in the P

site is moved to the amino acid on the

tRNA in the “A” site.• This is called “Elongation”

• 5.) The mRNA moves over one position.• The first tRNA now occupies the “E” site, the

second tRNA (with the growing amino acid chain)

now ocupies the “P” site and the “A site is open for

the next tRNA.

• 6.) The first tRNA is ejected from the “E”

site and goes into the cytoplasm to get

another amino acid.

Fig. 17-16b

P site (Peptidyl-tRNAbinding site) A site (Aminoacyl-

tRNA binding site)E site(Exit site)

mRNAbinding site

Largesubunit

Smallsubunit

(b) Schematic model showing binding sites

Next amino acidto be added topolypeptide chain

Amino end Growing polypeptide

mRNAtRNA

E P A

E

Codons

(c) Schematic model with mRNA and tRNA

5

3

Fig. 17-18-1

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

Fig. 17-18-2

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

Fig. 17-18-3

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

Fig. 17-18-4

Amino endof polypeptide

mRNA

5

3E

Psite

Asite

GTP

GDP

E

P A

E

P A

GDP

GTP

Ribosome ready fornext aminoacyl tRNA

E

P A

Termination of Translation

• Termination occurs when a stop codon in the mRNA

reaches the A site of the ribosome.

• The A site accepts a protein called a release factor.

• The release factor causes the addition of a water molecule

instead of an amino acid.

• This reaction releases the polypeptide, and the translation

assembly then comes apart.

Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

Fig. 17-19-1

Releasefactor

3

5

Stop codon(UAG, UAA, or UGA)

Fig. 17-19-2

Releasefactor

3

5

Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

Fig. 17-19-3

Releasefactor

3

5

Stop codon(UAG, UAA, or UGA)

5

3

2

Freepolypeptide

2 GDP

GTP

5

3

Animation

• Translation Animation

Mutations revisited

Types of mutations

• Point mutation:

– Examples:

• insertion,

• deletion,

• substitution,

• frameshift (results from insertion or deletion)

Effects of mutations

• 1.) Silent mutation• Has no effect, because the new codon codes for

the same amino acid as the old codon.

– Example: CUU, CUG, CUA, CUC all code for the amino

acid Leucine. So long as the 3rd nucleotide is the only

one that is changed, the effect is zero.

• 2.) Missense Mutation• The mutation causes a new codon that codes for a

new amino acid.

• This may have only a minor effect or it may result

in the production of a protein that is unable to form

into its proper 3-D shape and, therefore, is unable

to carry out its normal function.

• The hemoglobin protein that causes sickle-cell

disease is caused by a missense mutation.

• 3.) Non-sense Mutation• Occurs when the new codon is a stop codon.

DNA Organization

Background

• In eukaryotes, DNA is packaged with

proteins to form a matrix called chromatin

• The DNA is coiled around bundles of 8-9

histone proteins to form DNA-histone

complexes called nucleosomes.• Microscopically, nucleosomes look like beads on a

string.

Fig. 16-21a

DNA

double helix

(2 nm in diameter)

Nucleosome

(10 nm in diameter)

HistonesHistone tail

H1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Fig. 16-21b

30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber Looped domains (300-nm fiber)

Metaphase chromosome

• During cell division, DNA is compactly organized into chromosomes.

• When the cell is not dividing, there are 2 types of chromatin:– 1.) Euchromatin

– DNA is loosely bound to histones.

– DNA here is actively being transcribed.

– 2.) Heterochromatin– DNA is tightly bound to the histones.

– DNA is inactive in these regions.

What in the wide, wide world of

sports is a’ goin’ on here?!!?

Jumping genes?

Yep…

• Some DNA segments within genes are

able to move to new locations.

• This isn’t really a good thing….

• These transposible genetic elements,

called transposons (or jumping genes)

can move to a new location on the same

chromosome or to a different

chromosome.

History

• Discovered in the 1940s by Barbara McClintock.

– She also discovered that crossing over in meiosis

was a thing.

• A super important scientist most people have NEVER heard

of.

• She was studying maize.

– Findings:

• Parts of chromosomes can move randomly to

other locations on the chromosome, affecting

phenotypic expression.

• Genes can be turned on/off by environmental

factors.

• Genetic disorders can be reversed (in maize).

• Some transposons consist only of DNA that codes for an enzyme that enables it to be transported.

• Other transposons contain genes that invoke the replication of the transposon.

• After replication, the new transposon copy is transported to the new location.

• Wherever they are inserted, transposons have the effect of a mutation. – They may change the expression of a gene, turn on or off its

expression, or have no effect at all.

Stats:

• McClintock found that approx. 90% of

maize DNA consists of transposons.

– 44% in humans

In humans…

• Most common transposon is the Alu

sequence.

– 300 bp long.

– Occurs over 1 million different times in human

genome.

• Very common sequence (approx. 17% of total

genome).

– Insertions in Alu sequence in humans

generally have no effect b/c most of the

sequences occur in introns.

In humans…

• ACE gene (Angiotensin Converting

Enzyme)

– Comes in 2 varieties, one WITH an Alu

insertion, and one WITHOUT.

• Variation is linked to sporting performance:

– With = better at endurance events (distance running,

biking, distance swimming, etc).

– Without = better at strength/power events (weightlifting,

wrestling, etc).

In humans…

• Opsin gene duplication in Old World

Primates (including humans) is

hypothesized for the regaining of

trichromacy (3-color vision).

– Birds/fish have 4-color vision.

– Most other mammals are dichromatic.

The Molecular Genetics of

Viruses

Background

• Viruses are parasites of cells.

• Typical mode of infection:

1.) A typical virus penetrates a cell,

2.) it takes over the cell’s metabolic machinery,

3.) The virus (using the cell’s machinery) assembles

hundreds of new viruses that are copies of itself.

4.) Viruses then leave the cell (usually by destroying the

host cell) and infect other cells.

• Viruses are specific for the kinds of cells

they will parasitize.

– Some viruses only attack one type of cell

within a single host species.

– Others attack similar cells from a range of

closely related species.

– Bacteriophages, or phages, are viruses that

attack only bacteria.

Viral Structure

• Viruses consist of the following structures:

– A Nucleic Acid• Either DNA or RNA (not both)

• Contains the hereditary info of the virus

• May be double stranded (dsDNA or dsRNA) or single stranded (ssDNA or ssRNA)

– A Capsid• A protein coat that encloses the nucleic acid.

• Identical protein subunits, called capsomeres, assemble to form the capsid.

Viral Structure

• Some viruses have an envelope that

surrounds the capsid.

• The envelopes incorporate phospholipids

and proteins obtained from the cell

membrane of the host.

– Why would this be advantageous to the virus?

Viral StructureAnimal Virus StructureBacteriophage Structure

Enveloped Virus Structure

Types of Viruses

Viral Replication

• Two ways:

• 1.) Lytic Cycle• The virus penetrates the cell membrane of the host

cell and takes over the host cell.

• Once the viral particles have been replicated, the

host cell ruptures, releasing the viral particles.

• 2.) Lysogenic Cycle• viral DNA is temporarily incorporated into the host

cell DNA.

• A virus in this dormant stage is called a provirus

or if a bacteriophage a prophage.

• As the cell goes through mitosis, it copies the virus

as well.

• The virus remains inactive until some

environmental trigger causes the virus to begin the

destructive lytic cycle.

– Triggers = radiation, chemicals.

Retroviruses

• ssRNA viruses that use an enzyme called reverse transcriptase to make a DNA complement of their RNA.

• The DNA complement can then be transcribed immediately to manufacture mRNA (to make new viral proteins) or it can begin the lysogenic cycle.

• HIV works this way.

• Life cycle of HIV

The Molecular Genetics of

Bacteria

Background

• Bacteria have

– Cell walls

– Cell membranes

– Ribosomes

– DNA

• In a single circular

chromosome

• Bacteria lack

– Nucleus

– Specialized organelles

– Histones

Bacterial Chromosome

• Is often called a “naked chromosome”

because it lacks the histones and other

proteins associated with eukaryotic

chromosomes.

Bacterial Plasmids

• Bacteria contain short, circular DNA molecules outside the chromosome called plasmids.

• Plasmids carry genes that are beneficial but not normally vital for survival of the organism.

• Plasmids replicate independently of the chromosome.

• Some plasmids, called episomes, can become incorporated into the bacterial chromosome.

Genetic Variation in Bacteria

• Bacteria can alter their genome in 3 ways:

• 1.) Conjugation• DNA exchange between bacteria.

• A donor bacterium produces a tube, called a Sex

Pilus, that connects to another bacterium.

• The donor sends chromosomal or plasmid DNA to

the recipient through the pilus.

• In some cases, large portions of a donor’s

chromosome are sent, thus allowing recombination

with the recipient’s chromosome.

Genetic Variation in Bacteria

• Two plasmids in conjugation:

– 1.) F Plasmid– Contains the genes needed to produce pili (plural for

pilus).

– When the recipient bacterium receives the F plasmid, it

too can become a donor cell.

– 2.) R plasmid– Provide bacteria with antibiotic resistance.

Genetic Variation in Bacteria

• Bacteria can alter their genome in 3 ways:

• 2.) Transduction• Occurs when new DNA enters the bacterial

genome by way of a virus (bacteriophage).

• When a virus is assembled during the lytic cycle, it

is sometimes assembled with some bacterial DNA

in place of some viral DNA.

• When the new virus particles infect another cell,

the bacterial DNA they carry can recombine with

the resident DNA.

Genetic Variation in Bacteria

• Bacteria can alter their genome in 3 ways:

• 3.) Transformation• Occurs when bacteria absorb DNA from their

surroundings and incorporate it into their genome.

• Some bacteria have specialized proteins on their

cell membranes that allow this to occur.

Just for fun…

Regulation of Gene

Expression

Background

• Every cell in a human contains exactly the same sequences of DNA. Yet, some cells become muscle cells and other cells become nerve cells.

• One way that cells with identical DNA become different is by regulating gene expression through transcription of only selected genes.

• This is called “epigenetics”

Prokaryotic Gene Expression

• In prokaryotes, an operon is a unit of DNA

that controls gene transcription.

• Operons contain the following parts:

– 1.) Promoter

– 2.) Operator

– 3.) Structural Gene

– 4.) Regulatory Gene

• Promoter

– A sequence of DNA to which the RNA

polymerase attaches to begin transcription.

• Operator

– a region of DNA that can block the action of

RNA polymerase if the region is occupied by

a repressor protein.

• Structural Gene

– Contain DNA sequences that code for

several related enzymes that direct the

production of a particular end product.

• Regulatory Gene

– Is outside the operon region

– Produces repressor proteins and activator

proteins

• Repressor Protein

– Substances that occupy the operator region

and block the action of RNA polymerase.

• Activator Protein

– Assist the attachment of RNA polymerase to

the promotor region

Examples of Operons

• 1.) lac operon• In E. coli

• Controls the breakdown of lactose.

• Enzymes produced by the operon are inducible enzymes.

• 2.) trp operon • In E. coli

• Controls the production of the amino acid tryptophan

• Enzymes produced by the operon are repressible enzymes.

Operons

• Video 1

Eukaryotic Gene Expression

• 3 methods:

• 1.) Regulatory Proteins• Operate similarly to those in prokaryotes.

• Influence how readily the RNA pol will attach to the

promoter region.

• 2.) Nucleosome Packing• Influences whether a section of DNA will be

transcribed.

• DNA segments are tightly packed by methylationof the histones, which often makes the region un-transcribable.

– Barr Bodies are highly methylated

• DNA segments are loosely packed by acetylationof the histones, which allows the DNA to be transcribed.

• Heterochromatin & Euchromatin

• 3.) RNA Interference• Occurs when short interfering RNAs (siRNAs)

block mRNA transcription or translation.

– Under certain circumstances, an RNA molecule will fold

back and base pair with itself, forming dsRNA.

– An enzyme then cuts the dsRNA into short pieces, which

then base pair to complimentary DNA regions,

preventing further transcription of the gene.

– Sometimes the siRNAs will bind to existing mRNA

strands, thereby preventing ribosomes from binding to

the mRNA. This effectively inactivates the mRNA.

• 4.) Alternative RNA Splicing• Certain codons are copied from a gene when other

codons in same gene are not.

• This allows the same gene to code for many

different proteins.

• We’ve discussed this before.

• 5.) Protein degradation • As proteins age, they lose functionality due to bond

interferences.

• Proteins that need to be recycled due to non-

function are marked for destruction with a protein

called ubiquitin (so called because it is ubiquitous,

found in all eukaryotic cells).