Genes and Protein Synthesis

Chapter 7

One Gene-One Polypeptide Hypothesis• DNA contains all of

our hereditary information

• Genes are located in our DNA

• ~25,000 genes in our DNA (46 chromosomes)

• Each Gene codes for a specific polypeptide

Main Idea• Central Dogma– Francis Crick (1956)

Overall Process• Transcription – DNA to RNA

• Translation – Assembly of

amino acids into polypeptide

– Using RNA

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

Key Terms • RNA transcription– Initiation,

Elongation, Termination

• TATA box • Introns, Exons• mRNA, tRNA, rRNA• Translation • Ribosome• Codon• Amino Acids• Polypeptide

DNA RNA

Double stranded Single stranded

Adenine pairs with Thymine Adenine pairs with Uracil

Guanine pairs with Cytosine Guanine pairs with Cytosine

Deoxyribose sugar Ribose sugar

DNA to Protein • Protein is

made of amino acid sequences

• 20 amino acids

• How does DNA code for amino acid?

DNA molecule

Gene 1

Gene 2

Gene 3

DNA strand

TRANSCRIPTION

RNA

Polypeptide

TRANSLATIONCodon

Amino acid

Genetic Code• Codon– Three letter code– 5’ to 3’ order– Start codon – Stop codon

• AA are represented by more than one codon

• 61 codons that specify AA

Amino acids • Abbreviated– Three letters

Transcription • DNA to RNA• Occurs in nucleus • Three process– Initiation – Elongation – Termination

RNA polymerase

DNA of gene

PromoterDNA Terminator

DNAInitiation

Elongation

TerminationGrowingRNA

RNApolymerase

Completed RNA

Initiation• RNA polymerase binds to DNA• Binds at promoter region

– TATA box• RNA polymerase unwinds DNA• Transcription unit

– Part of gene that is transcribed• Transcription factors bind to

specific regions of promoter • Provide a substrate for RNA

polymerase to bind beginning transcription

• Forms transcription initiation complex

Elongation • RNA molecule is

built– RNA polymerase

• Primer not needed• 5’ to 3’ direction • Template strand is

copied– 3’ to 5’ DNA

• Coding strand– DNA strand that is

not copied• Produces mRNA

– Messenger RNA • DNA double helix

reforms

Termination • RNA polymerase recognizes a termination sequence

– AAAAAAA (polyadenylation)• Nuclear proteins bind to string of UUUUUU on RNA• mRNA molecule releases from template strand

Post-Transcriptional Modifications• Pre-mRNA

undergoes modifications before it leaves the nucleus

• Poly(A) tail– Poly-A polymerase– Protects from RNA

digesting enzymes in cytosol

• 5’ cap– 7 G’s– Initial attachment

site for mRNA’s to ribosomes

• Removal of introns

Splicing the pre-mRNA• DNA comprised of – Exons • sequence of DNA or

RNA that codes for a gene

– Introns • non-coding

sequence of DNA or RNA

• Spliceosome– Enzyme that

removes introns from mRNA

Splicing Process• Spliceosome contains a handful of small

ribonucleoproteins– snRNP’s (snurps)

• snRNP’s bind to specific regions on introns

Alternative Splicing• Increases number and variety of proteins

encoded by a single gene• ~25,000 genes produce ~100,000 proteins

Translation• mRNA to protein • Ribosomes read

codons • tRNA assists

ribosome to assemble amino acids into polypeptide chain

• Takes place in cytoplasm

tRNA• Contains – triplet anticodon – amino acid

attachment site • Are there 61

tRNA’s to read 61 codons?

tRNA: Wobble Hypothesis • First two nucleotides of

codon for a specific AA is always precise

• Flexibility with third nucleotide

• Aminoacylation– process of adding an AA

to a tRNA – Forming aminoacyl-

tRNA molecule – Catalyzed by 20

different aminoacyl-tRNA synthetase enzymes

Ribosomes• Translate mRNA chains into amino acids• Made up of two different sized parts – Ribosomal subunits (rRNA)

• Ribosomes bring together mRNA with aminoacyl-tRNAs

• Three sites– A site - aminoacyl– P site – peptidyl– E site - exit

1 Codon recognition

Amino acid

Anticodon

AsiteP site

Polypeptide

2 Peptide bond formation

3 Translocation

Newpeptidebond

mRNAmovement

mRNA

Stopcodon

Translation process • Three stages– Initiation – Elongation – Termination

Initiation• Ribosomal subunits associate with mRNA • Met-tRNA (methionine)

– Forms complex with ribosomal subunits• Complex binds to 5’cap and scans for start codon (AUG) (scanning)• Large ribosomal subunit binds to complete ribosome • Met-tRNA is in P-site

Reading frame is established to correctly read codons

Elongation

• Amino acids are added to grow a polypeptide chain

• A, P, and E sites operate

• 4 Steps

Termination• A site arrives at a stop codon on mRNA – UAA, UAG, UGA

• Protein release factor binds to A site releasing polypeptide chain

• Ribosomal subunits, tRNA release and detach from mRNA

ba

Red object = ?

What molecules are present in this photo?

POLYSOME

Review • What is a gene?• Where is it

located?• What is the main

function of a gene?• Do we need our

genes “on” all the time?

• How do we turn genes “on” or “off”?

Regulating Gene Expression• Proteins are not

required by all cells at all times

• Regulated• Eukaryotes – 4 ways– Transcriptional (as mRNA

is being synthesized)– Post-transcriptional (as

mRNA is being processed)

– Translational (as proteins are made)

– Post-translational (after protein has been made)

Transcriptional regulation• Most common• DNA wrapped around histones keep gene promoters

inactive• Activator molecule is used (2 ways)– Signals a protein remodelling complex which loosen the

histones exposing promoter– Signals an enzyme that adds an acetyl group to histones

exposing promoter region

Transcriptional regulation• Methylation– Methyl groups are added to the cytosine bases in the

promoter of a gene (transcription initiation complex) – Inhibits transcription – silencing– Genes are placed “on hold” until they are needed– E.g. hemoglobin

Post transcriptional regulation• Pre-mRNA processing

– Alternative splicing• Rate of mRNA

degradation– Masking proteins used to

degrade mRNA – Translation does not occur

• Embryonic development

• Hormones – Casein – milk protein in

mammary gland– When casein is needed,

prolactin is produced extending lifespan of casein mRNA

Translational regulation• Occurs during

protein synthesis by a ribosome

• Changes in length of poly(A) tail– Enzymes add or

delete adenines – Increases or

decreases time required to translate mRNA into protein

– Environmental cues

Post-Translational Regulation• Processing

– Removes sections of protein to make it active

– Cell regulates this process (hormones)

• Chemical modification– Chemical groups are

added or deleted – Puts the protein “on hold”

• Degradation– Proteins tagged with

ubiquitin are degraded – Amino acids are recycled

for protein synthesis

Cancer• Lack regulatory mechanisms • Mutations in genetic code

(mutagens)– Probability increases over lifetime– Radiation, smoking, chemicals

• Mutations are passed on to daughter cells – Can lead to a mass of

undifferentiated cells (tumor)– Benign and malignant

• Oncogenes– Mutated genes that once served

to stimulate cell growth– Cause undifferentiated cell

division

Genetic Mutations• Positive and negative – Natural selection –

evolution – Cancer –death

• Small-Scale – single base pair– Point mutations • Substitution,

insertion/deletion, inversion

• Large-Scale – multiple base pairs

Small-Scale Mutations• Four groups – Missense, nonsense, silent, frameshift• Lactose, sickle cell anemia

– SNPs – single nucleotide polymorphisms• Caused by point mutations

Missense mutation• Change of a single base pair or group of base pairs• Results in the code for a different amino acid • Protein will have different sequence and structure

and may be non-functional or function differently

Nonsense mutation• Change in single base pair or group of base pairs • Results in premature stop codon • Protein will not be able to function

Silent Mutation• Change in one or more base pairs• Does not affect functioning of a gene• Mutated DNA sequence codes for same amino acid • Protein is not altered

Frameshift mutation• One or more nucleotides are inserted/deleted from a DNA

sequence• Reading frame of codons shifts resulting in multiple missense

and/or nonsense effects• Any deletion or insertion of base pairs in multiples of 3 does

not cause frameshift

Large-scale mutations • Multiple nucleotides,

entire genes, whole regions of chromosomes

Large-scale mutations • Amplification – gene

duplication– Entire genes are copied to

multiple regions of chromosomes

Large-scale mutations • Large-scale deletions – Entire coding regions of DNA are removed • Muscular Dystrophy

Large-scale mutations • Chromosomal translocation– Entire genes or groups of genes are moved from one

chromosome to another – Enhance, disrupt expression of gene

Large-scale mutations • Inversion– Portion of a DNA molecule reverses its direction in the

genome– No direct result but reversal could occur in the middle of

a coding sequence compromising the gene

Large-scale mutations • Trinucleotide repeat expansion– Increases number of repeats in genetic code – CAG CAG CAG CAG CAG CAG CAG CAG • Huntingtons disease

Causes of genetic mutations• Spontaneous mutations– Inaccurate DNA replication

• Induced mutations – Caused by environmental agent – mutagen – Directly alter DNA – entering cell nucleus – Chemicals, radiation

Chemical Mutagens • Modify individual

nucleotides– Nucleotides

resemble other base pairs

– Confuses replication machinery – inaccurate copying • Nitrous acid

• Mimicking DNA nucleotides – Ethidium bromide –

insert itself into DNA

Radiation - Low energy • UV B rays • Non-homologous end joining– Bonds form between adjacent nucleotides along DNA

strand – Form kinks in backbone – Skin cancer

Radiation – high energy • Ionizing radiation – x-ray, gamma rays • Strip molecules of electrons • Break bonds within DNA– Delete portions of chromosomes

• Development of tumors

Genomes and Gene organization• Human Body– 22 autosomal chromosomes– 1 pair of each sex chromosome (XX, YY)

Genomes and Gene organization• Components– VNTR’s–variable number tandem repeats (microsatellites)• Sequences of long repeating base pairs• TAGTAGTAGTAGTAG

– LINEs – long interspersed nuclear elements – SINEs – short interspersed nuclear elements – Transposons – small sequences of DNA that move about

the genome and insert themselves into different chromosomes

– Pseudogene – code is similar to gene but is unable to code for protein

Viruses• Not alive but can replicate themselves• Contain– DNA or RNA– Capsid – protein coat– Envelope – cell membrane

Virus • 4000 species of virus have been classified

HIV RNA Replication (Retrovirus) • Reverse

transcriptase to turn RNA into DNA

• Integrase incorporates into our genetic code

• Uses cells parts to make protein parts from mRNA

• genomic RNA

Influenza A• Viral RNA

replicated and transcribed for protein synthesis

Virus as Vectors• Transduction– Using a virus vector to insert DNA into a cell or

bacterium