8/13/2016 Central Dogma of Biology Chapter 17 Flow of genetic - - PDF document

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Chapter 17 PROTEIN SYNTHESIS: FROM GENE TO PROTEIN Central Dogma of Biology

Flow of genetic information:

RNA (ribonucleic acid)

DIFFERENCES DNA RNA

deoxyribose sugar double strand bases A,T,C,G found in: nucleus, mitochondria, chloroplasts ribose sugar single strand bases A,U,C,G found in: nucleus, cytosol, ribososomes many functions

RNA has many functions in the cell.

• 1. pre-mRNA: precursor to mRNA, newly transcribed and not edited
• 2. mRNA: carries the code from DNA that specifies amino acids
• 3. tRNA: carries a specific amino acid (anticodon)to ribosome
• 4. rRNA: makes up 60% of the ribosome; site of protein synthesis
• 5. snRNA: small nuclear RNA; part of a spliceosome (in RNA splicing)
• 6. srpRNA: a signal recognition particle that binds to signal peptides
• 7. RNAi: interference RNA; a regulatory molecule
• 8. ribozyme: RNA molecule that functions as an enzyme

3 types RNA directly involved in making proteins

• 1. messenger RNA (mRNA)

single uncoiled long strand

• transmits DNA info

during protein synthesis

• serves as template to

assemble amino acids

• 2. transfer RNA (t RNA)
• carries amino acids to

ribosome

• 3. ribosomal RNA (r RNA)

makes up large part (2/3) of ribosome

• globular

PROTEIN SYNTHESIS/GENE EXPRESSION

Formation of proteins using information coded on DNA and carried out by RNA.

• ne gene = one RNA molecule

Gene: region of DNA whose final product is either a polypeptide or RNA molecule A gene is expressed when protein synthesis is occurring.

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The Genetic Code

How is information necessary for creating proteins encoded in the RNA? The genetic code from DNA is transcribed onto m RNA by Codons. For each gene, one DNA strand is the template strand mRNA (5’  3’) complementary to template mRNA triplets (codons) code for amino acids in polypeptide chain

Code word/Codon (triplet):

specific group of 3 successive bases on DNA and mRNA

• codes for a specific amino acid to be placed on

the protein chain

• 20 biological amino acids, but more than 20 codons

Like “genetic words” DNA code words: ACT, GCA, TTA RNA codons: UGA, CGU, AAU How many combinations of code words can we make from 4 bases? 64 different codon combinations ( 43 = 64) ** each code word always codes for same amino acid** Redundancy: 1+ codons code for each of 20 AAs Reading frame: groups of 3 must be read in correct groupings This code is universal: all life forms use the same code

Ala: Alanine Cys: Cysteine Asp: Aspartic acid Glu: Glutamic acid Phe: Phenylalanine Gly: Glycine His: Histidine Ile: Isoleucine Lys: Lysine Leu: Leucine Met: Methionine Asn: Asparagine Pro: Proline Gln: Glutamine Arg: Arginine Ser: Serine Thr: Threonine Val: Valine Trp: Tryptophane Tyr: Tyrosisne

It is possible for non-Watson-Crick base pairing to occur at the third codon position. This has phenomenon been termed the wobble hypothesis.

How do these code words affect protein synthesis?

Order of code words codes for Order of amino acids codes for Specific type of protein

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BUILDING OF PROTEINS

• DNA unzips
• original strand of DNA acts as template for m RNA

(only one strand of DNA molecule needed for transcription) euchromatin: uncoiled areas of DNA, active site of transcription antisense /non-template strand: non coding strand of DNA sense/template strand: coding strand of DNA

2 Stages of Protein Synthesis Stages of Protein Synthesis

• I. Transcription (nucleus)

Process where mRNA is produced from DNA Transcription unit: stretch of DNA that codes for a polypeptide or RNA

Transcription

• 1. Initiation (prokaryotes)

RNA polymerase binds directly to promoter in DNA

• promoter : region of DNA that initiates transcription of a

particular gene.

• located near the transcription start sites of

genes on sense strand

• located upstream on the DNA

(towards the 5’ region of the antisense strand) **can be about 100–1000 base pairs long**

nontemplate strand template strand

Steps of transcription

• 1. Initiation (eukaryotes)
• A. The promoter site on the

DNA contains a sequence called a TATA box

• TATAAAA
• recognized by RNA

polymerase ll

• can be up to 25 bases

upstream from point of transcription

• B. Transcription factor proteins

and RNA polymerase ll bind to promoter section of DNA and unwinds the part of the DNA to be transcribed. (this is the structural gene: codes for a single protein)

• 2. Elongation
• A. RNA polymerase reads DNA template strand
• B. Complementary nucleotides are added to the 3'

end of RNA using information in DNA as instructions **Polymerases always work from the 3' to the 5' end of the coding strand

• f DNA (template); thus the

antiparallel structure it is forming is going from the 5' to 3’ direction.

• C. As RNA polymerase moves, it untwists

DNA, then rewinds it after mRNA is made

• D. Once RNA nucleotides are attached to

DNA chain, codons are in proper order

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• 3. Termination

A. RNA polymerase transcribes a terminator sequence in DNA B. mRNA and polymerase detach. * prokaryotes- mRNA ready to use * eukaryotes- pre-mRNA, will undergo further modifications transcription animation RNA Processing Occurs After Transcription

• 1. Alteration of pre-mRNA ends
• 5’ cap (modified guanine) and 3’poly-A tail (50-520 A’s) are

added

• functions:
• help export mature mRNA from nucleus
• protect mRNA from enzyme degradation
• help ribosomes attach to mRNA

RNA Processing Occurs After Transcription

• 2. RNA Splicing
• Pre-mRNA has introns (noncoding sequences) and

exons (codes for amino acids)

• Splicing- introns cut out, exons joined together
• Once RNA is spliced it can move out of the nucleus to be

translated RNA Splicing, cont. snRNPs small nuclear ribonucleoproteins snRNP = snRNA + protein Pronounced “snurps” Recognize splice sites snRNPs join with other proteins to form a spliceosome Spliceosomes catalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme

RNA splicing animation

Importance of Introns

• Functions not known for most introns
• Some regulate gene expression
• Alternative RNA Splicing: produce

different combinations of exons

• Depends which segments are

treated as exons during splicing

• One gene can make more than one

polypeptide

• 20,000 genes  100,000

polypeptides Changes in gene expression may confer an evolutionary advantage

Prokaryotic vs Eukaryotic Gene Expression

Prokaryotes

• Transcription and translation both

in cytoplasm

• RNA poly binds directly to

promoter

• No introns
• No RNA splicing

Eukaryotes

• Transcription in nucleus;

translation in cytoplasm

• DNA in nucleus, RNA travels

in/out nucleus

• RNA poly binds to TATA box &

transcription factors

• RNA contains introns
• RNA splicing

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Translation (in cytoplasm at ribosome)

• process whereby protein is synthesized

from mRNA

• newly synthesized mRNA moves from nucleus to

ribosome in cytoplasm

• gene has 3x more nucleotides than the protein it

makes Ex: 100 a.a. = 300 nucleotides

• components of translation
• mRNA- message
• tRNA- interpreter
• ribosome- site of translation

Transfer RNA (t RNA)

• function: transfers amino

acids to ribosome

• 20 types – one for each

amino acid (specific)

• structure (cloverleaf)
• found in cytosol

Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid Ribosomes

• made in nucleolus
• 2 subunits make up

ribosome

• about 2/3 is r-RNA and

1/3 is protein

• smaller subunit has

binding site for mRNA

• normally apart in

cytoplasm, come together during protein synthesis

Steps of translation

• 1. Initiation
• A. 5’ end of m RNA binds to small subunit
• B. initiator tRNA carrying Met attaches to P site
• C. large subunit attaches (ribosome ready for protein synthesis)
• sites: locations on ribosome where tRNA anticodons

attach P (peptidyl) site- holds aa chain A (aminoacyl) site- holds aa to be added E (exit) site- exit for tRNA * start codon (AUG) will be at the site

• n mRNA where this occurs

** anticodon on first tRNA will always be UAC, amino acid 1 will always be methionine

• 2. Elongation
• A. t-RNA with a specific anticodon binds a

specific amino acid. This happens for several t-RNAs and proper corresponding amino acids in the cytoplasm. ATP: energy source used to bind the amino acid to the t-RNA. Aminoacyl-tRNA synthase: enzyme that does the binding.

• B. First tRNA binds to P site, second tRNA

binds to A site (anticodons are complementary to mRNA codons)

• C. Peptidyl transferase reaction occurs:

#1 a.a. joins to #2 a.a.

• D. Ribosome moves down mRNA and first

tRNA is released to be used over again

• translocation: movement of

ribosome down mRNA

• E. Amino acids continue to be added to

protein chain thru same mechanism

• peptidyl synthase: enzyme that

joins a.a. together

• 3. Termination
• A. stop codon is reached (UAA, UGA, or UAG).
• B. release factor binds to stop codon and polypeptide is released
• C. subunits dissociate (can be used over again)
• D. protein is released into cell
• E. mRNA is broken down by cell (not be used again – only once)
• F. tRNA is released into cell (used over again)

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Speed of Translation

• process occurs from minutes

to hours in an organism

• Polyribosomes: strings of

ribosomes that can translate many copies of a polypeptide very quickly

• During synthesis, polypeptide

chain coils and folds spontaneously

Protein synthesis animation Protein synthesis animation 2

Where to proteins go once synthesized?

Free ribosomes (floating in cytosol)

• make proteins that stay in cytosol to perform functions

Bound ribosomes (attached to ER)

• make proteins for secretion
• Use signal peptide to target their location
• make proteins of the endomembrane system
• nuclear envelope, ER, Golgi, lysosomes, vacuoles,

plasma membrane)

Signal Mechanism

Signal peptide: 20 AA at leading end of polypeptide determines destination Signal-recognition particle (SRP): brings ribosome to ER

Mutations

• Changes in genetic code of a cell
• Source of new genes and diversity of genes among organisms
• Mutagen: substance that causes mutations
• Radiation, chemicals, viruses
• 1. Large scale (chromosome)
• Involve large segments of chromosome
• Cause disorders or death
• Types: duplications, large deletions, translocation, inversion,

non-disjunction

• 2. Small scale
• Single nucleotide-pair substitutions
• Nucleotide-pair insertions or deletions (one or more pairs)

Large Scale Chromosome Mutations Review Small Scale Mutations Point mutations: alter 1 base pair of a gene

• 1. Base-pair substitutions – replace 1 with another
• Silent: no change in amino acid due to redundancy
• Missense: change one amino acid into another
• Nonsense: change into stop codon, results in

shorter non-functional polypeptide

• 2. Frameshift: alters reading frame of RNA

causes non-functional proteins

• Insertions: addition of nucleotide/s
• Deletions: removal of nucleotide/s

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Base Pair Substitutions

Substitution: Silent (no effect)

Base Pair Substitutions

Substitution- Missense (change of one amino acid to another) Sickle Cell Anemia: point mutation

Base Pair Substitutions

Substitution: Nonsense (change into stop codon)

Frameshift

Insertion: shifts frame to right

Frameshift

Deletion: shifts frame to left, premature termination

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Summary of Protein Synthesis