Molecular Biology: From DNA to Protein (Part II) Transcription • Purpose and sub-cellular compartment. • Steps of transcription and the bio-molecules involved. • Terminology: promoter and terminator DNA sequences RNA Processing in eukaryotic cells Genetic code • The language of nucleic acids: letters and words (nucleotides and codons) • Initiation and termination codons of translation Mutation • Definition, sources, impact the protein product Translation or protein synthesis • Purpose and sub-cellular compartment and its three steps. • The interpreter the language of nucleic acids into the language of proteins Use the genetic code table to translate a sequence of codons into a sequence of

amino acids and the reverse as well as the sequence of normal and mutated proteins

How DNA directs the functions of the cell?

• RNA Structure • Transcription process, steps, and molecular participants • Translation Process and Steps and molecular participants

Key Hydrogen atom Carbon atom Nitrogen atom Oxygen atom Phosphorus atom

Nitrogenous base (A, G, C, or

U) Phosphate group

O

O–

O O CH2

H C

C

C

C N

C

N

H

H

O

O

C

O

O

H

C H H

OH

C

H

Uracil (U)

Sugar (ribose)

P

Transcription of a gene 1. DNA: double –stranded 2. Enzyme: RNA Polymerase 3. Monomers: RNA ribonucleotides 4. Steps:

Initiation- Starts at promoter sequence

Elongation Termination- Stops at termination

sequence

RNA polymerase

DNA of gene

Promoter DNA

Terminator DNA

Growing RNA

Completed RNA RNA polymerase

Figure 10.9B

1 Initiation

2 Elongation

3 Termination

Transcription

Transcription Animation

Template and coding strands of DNA

- The two strands of DNA have complementary sequences - (template/and coding strand)

- RNA transcripts are single stranded and are made 5’ 3’

- Only one DNA strand, the template strand is copied into RNA

If given the sequence of one of the strands, predict the sequences

of the other two using the base-pairing rule

Activity Which is the sequence of the mRNA transcript of the following DNA sequence? DNA: 5'...CCATGGATCTTAGCA...3' (template or strand)

3'...GGTACCTAGAATCGT...5‘ (coding Strand)

– RNA polymerase • unwinds the two DNA

strands • Uses one strand as a

template strand • Polymerizes RNA nucleotides

against the template strand following the base pairing rules

– Single-stranded messenger RNA (mRNA) peels away from the template strand

– The DNA strands rejoin

(a) Bacterial cell

TRANSCRIPTION DNA

mRNA

TRANSLATION Ribosome

Polypeptide

(b) Eukaryotic cell

TRANSCRIPTION

Nuclear envelope

DNA

Pre-mRNA RNA PROCESSING

mRNA

TRANSLATION Ribosome

Polypeptide

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RNA processing protects RNA

Section 7.4

A “cap” structure is added to the 5’ end of mRNA.

A poly-A tail structure is added to the 3’ end of mRNA.

The cap and tail protect mRNA from degradation.

Figure 7.11

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RNA processing removes extra sequences

Section 7.4

Introns are sequences in genes that are not used for producing a protein.

Exons are the sequences that specify amino acids.

Introns are removed from the mRNA.

Figure 7.11

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Processed RNA is ready for translation

Section 7.4

The processed RNA is now a functional molecule.

When processing is complete, it leaves the nucleus.

Onward to translation!

Figure 7.11

Post-transcriptional modifications of eukaryotic pre-mRNA or primary transcript

- 5’ cap: protection against nucleases degradation and binding to ribosomes - 3’ poly A tail: transport to cytoplasm, efficient

translation, turn over rate of the mRNA - RNA splicing to connect the exons after removal

of introns Spliceosomes http://highered.mcgraw-

hill.com/sites/0072437316/student_view0/chapter15/animations.html#

• Proteins discrete structural and functional regions called domains.

• Different exons code for different domains of a protein.

THE FLOW OF GENETIC INFORMATION FROM DNA TO RNA TO PROTEIN

• The information carried by sequence of DNA bases constitutes an organism’s genotype

• The DNA genotype is expressed as proteins, which provide the molecular basis for the phenotype

Genetic information written in a code that is translated into amino acid sequences

The “words” of the DNA “language” are triplets of bases (3 bases long) called codons

Each codons in a gene specify one amino acid

sequence of the polypeptide

Protein synthesis Translation is the RNA–directed synthesis of a

polypeptide Translation of the language of nucleic acids into

the language of proteins (amino acids) One codon ------ one amino acid

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand (template)

3′

TRANSCRIPTION

Codon

mRNA

TRANSLATION

Protein

Amino acid

3′ 5′

5′

From the language of DNA to the language of proteins

The genetic code is the Rosetta stone of life Nearly all organisms use exactly the same genetic code

Figure 10.8A

UUC UGU UGC UGA Stop

Met or start

Phe

Leu

Leu

Ile

Val Ala

Thr

Pro

Ser

Asn

Lys

His

Gln

Asp

Glu

Ser

Arg

Arg

Gly

Cys Tyr

G

A

C

U

U C A G

Second base

Firs

t bas

e

UUA

UUU

CUC CUU

CUG CUA

AUC AUU

AUG

AUA

GUC GUU

GUG

GUA

UCC UCU

UCG

UCA

CCC CCU

CCG CCA

ACC ACU

ACC ACA

GCC GCU

GCG

GCA

UAC UAU

UAG Stop

UAA Stop

CAC CAU

CAG CAA

AAC AAU

AAG AAA

GAC GAU

GAG

GAA

UGG Trp

CGC CGU

CGG CGA

AGC AGU

AGG AGA

GGC GGU

GGG

GGA

U

C

A

G

U

C

A

G

U

C

A

G U

C

A

G

In-class activity/Genetic code Use the genetic code table to answer the following questions 1. How many codons are there for leu (leucine)? 2. How many codons are there for Met (Methionine)? 3. How many codons are there for Phe (phenylalanine)? Draw a conclusion about the number of codons for amino acids. 4. How many “stop” codons are there? Answer the following questions using this genetic code: 5’-AUGACCCCUUUGUUAUACUAA-3’ 5. How long is this message in nucleotides? 6. Is this the information present in DNA or in mRNA? Explain your answer . 7. Write down the sequence of amino acids coded for by the above stretch of nucleotides. how long is this polypeptide? .

Mutation/ In- class

1. For amino acids with redundant codons, which nucleotide position(s) are always the same, i.e conserved? (marked next to the genetic code table) . 2. Which amino acid does UUA code for? . 3. Does a mutation that changes the codon UUA into a UUG change the amino acid sequence at the protein level? . 4. Does a DNA mutation changing the codon UUA into a UCA change the amino acid sequence at the protein level? . 5. What impact would a change of the codon UUA into a UAA have at the translational level? . In Question 3, the mutation is a . In Question 4, the mutation is a . In Question 5, the mutation is a .

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Nucleotide substitutions cause small changes in protein structure

Section 7.7

Wild type = original nucleotide sequence

Substitution = changed nucleotide(s)

Table 7.2

T A B L E 7.2 Types of Mutations Type Illustration Original Sentence

THE ONE BIG FLY HAD ONE RED EYE

Substitution (missense)

THQ ONE BIG FLY HAD ONE RED EYE

Nonsense THE ONE BIG Insertion THE ONE BIG WET FLY HAD ONE RED EYE Insertion (frameshift)

THE ONE QBI GFL YHA DON ERE DEY

Deletion THE ONE BIG HAD ONE RED EYE Expanding repeat

Generation 1: THE ONE BIG FLY HAD ONE RED EYE Generation 2: THE ONE BIG FLY FLY FLY HAD ONE RED EYE Generation 3: THE ONE BIG FLY FLY FLY FLY FLY FLY HAD ONE RED EYE

Only one codon is altered, so only one amino acid in the protein will be affected.

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Some mutations cause disease

Section 7.7

A single base substitution in a hemoglobin gene causes blood cells to form abnormally, leading to sickle cell disease.

Figure 7.21

Normal red blood cells

Sickled red blood cells

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“Frameshift” mutations cause large changes in protein structure

Section 7.7

Insertion of one nucleotide changes every codon after the insertion.

Table 7.2

T A B L E 7.2 Types of Mutations Type Illustration Original Sentence

THE ONE BIG FLY HAD ONE RED EYE

Substitution (missense)

THQ ONE BIG FLY HAD ONE RED EYE

Nonsense THE ONE BIG Insertion THE ONE BIG WET FLY HAD ONE RED EYE Insertion (frameshift)

THE ONE QBI GFL YHA DON ERE DEY

Deletion THE ONE BIG HAD ONE RED EYE Expanding repeat

Generation 1: THE ONE BIG FLY HAD ONE RED EYE Generation 2: THE ONE BIG FLY FLY FLY HAD ONE RED EYE Generation 3: THE ONE BIG FLY FLY FLY FLY FLY FLY HAD ONE RED EYE

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Frameshifts affect multiple amino acids

Section 7.7 Figure 7.22

Original DNA sequence

One base added (frameshift mutation)

Two bases added (frameshift mutation)

Three bases added (reading frame not disrupted)

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Mutations create genetic variety

Section 7.7

Mutations are not always harmful.

Mutations create different versions of alleles, which are alternative versions of the same gene.

Genetic variation is important for evolution.

Plant breeders even induce mutations to create new varieties of plants.

Figure 7.23

(a): ©Erich Schlegal/Dallas Morning News/Corbis; (b): ©Pallava Bagla/Corbis; (c): ©Scott Olson/Getty Images

Translation • Takes place in cytoplasm • Involves: Ribosomes: Two subunits (each made of

many proteins & r-RNA) mRNA t-RNA (interpreter) 20 amino acids

A codon start codon within the mRNA message marks the translation initiation and a stop codon marks the end of translation

Start of genetic message

End

Figure 10.13A

Transfer RNA molecules serve as interpreters during translation

- Each tRNA molecule is a folded molecule bearing a base triplet called an anticodon on one end - A specific amino acid is attached to the other end

Amino acid attachment site

Anticodon

Amino acid attachment site

Hydrogen bond

RNA polynucleotide chain

Anticodon Figure 10.11A

Steps of protein Synthesis 1. Initiation a. binding of mRNA to small ribosomal subunit b. binding of Met-tRNA to AUG on mRNA c. Binding of large ribosomal subunit

2. Elongation a. binding of a second tRNA to next codon b. formation of peptide bond between Met and the second

amino acid c. sliding of ribosome by one codon Amino acids are added to the growing polypeptide chain until a

stop codon is reached.

3. Termination Disassembly of the protein synthesis machinery

mRNA, a specific tRNA, and the ribosome subunits assemble during initiation

Met Met

Initiator tRNA

1 2 mRNA Small ribosomal

subunit

Start codon

Large ribosomal subunit

A site U A C A U C

A U G A U G

P site

Figure 10.13B

Elongation steps

Polypeptide

P site

mRNA Codons

mRNA movement

Stop codon

New Peptide bond

Anticodon

Amino acid

A site

Figure 10.14

1 Codon recognition

2 Peptide bond formation

3 Translocation

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Translation is efficient

Section 7.5

Multiple ribosomes attach to an mRNA molecule simultaneously, so the cell can make many molecules of protein

all at once.

Figure 7.16

Review: The flow of genetic information in the cell is DNA→RNA→protein

The sequence of codons in DNA, via the sequence of codons in mRNA spells out the primary structure of a polypeptide

From Gene to Protein

http://www.wiley.com/college/test/0471787159/biology_basics/animations/fromGeneToProtein

.swf (highly recommended)

http://learn.genetics.utah.edu/content/molecules/transcribe/