Protein Synthesis
Protein Synthesis Transcription
- Proteins
- “Workhorse” molecule
- Blueprint for proteins is DNA
- Responsible for phenotype
- Gene -- part of DNA that codes for a particular polypeptide chain
- Use of proteins
- Enzymes (catalase)
- Structure (silk, hair, nails)
- Antibodies
- Movement (muscle, flagella)
- Hormones (insulin)
- Carry gasses (hemoglobin)
- Storage of amino acids (albumin)
- History
- 1909, Archibald Gerrod -- thought genes determine phenotype through defective enzymes controlling biochemical pathways
- First to connect human disorder with mendel’s laws of inheritance
- Proposed idea that diseases came about through metabolic route
- Studied alkaptonuria and deduced it was a recessive disorder
- Beadle and Tatum -- established link between genes and enzymes by studying bread mold; proposed a direct link between genes and enzymes (“one gene, one enzyme” theory which is wrong)
CONCLUSION:
- One gene produces one enzyme
- Was later modified to one gene produces one protein
- Then modified to one gene produces one polypeptide chain
- TRUTH is that one gene codes for an RNA molecule
- Overview
- DNA is in nucleus
- Proteins are made in cytoplasm
- RNA is intermediate between DNA code and actual synthesis of a protein
1. Transcription -- DNA nucleotides from a gene are used as a template to make RNA
2. Translation -- RNA template is used with ribosomes and tRNA with attached amino acids to make a polypeptide chain
RNA | DNA |
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- Types of RNA
- mRNA -- carries message from DNA gene to cytoplasm; determines sequence of amino acids for protein
- tRNA -- brings correct amino acid to ribosome & mRNA in translation
- rRNA -- in ribosome; “connects” tRNA to mRNA
- snRNA -- on spliceosomes; removes introns
- SRP RNA -- part of signal recognition particle used to bring translating ribosome to ER and threads emerging polypeptide chain into lumen of ER (helps to make proteins that are meant to travel outside of the cell)
- Genetic Code
- Codon -- codes for amino acid; a triplet of DNA nucleotides
- 64 codons (61 for amino acids, 3 stop codons)
- More than one codon codes for same amino acid (redundancy)
- Start codon is methionine
- Marshall Nirenberg and Heinrich Matthael determined 1st codon for amino acid
- Found that UUU coded for phenylalanine
- created mRNA entirely of uracil and added it to test tube with materials for protein synthesis (amino acids, ribosomes, RNA polymerase, etc)
- Code is universal -- all cells use same code and eukaryotic gene can be expressed in prokaryotic cells
- EXCEPTIONS
- Paramecia -- UAA and UAG code for glutamine instead of STOP
- Animal mitochondria -- AUA used for methionine instead of isoleucine
- Vertebrate mitochondrial -- AGA and AGG as stop codons
- TRANSCRIPTION OVERVIEW
- Transcription -- RNA synthesis from DNA template
- 1) initiation
- Promoter region -- region prior to beginning of a gene where RNA polymerase attaches; determines which side of gene will be transcribed
- In prokaryote, RNA polymerase attaches directly to region
- One type of RNA polymerase
- In Eukaryotic, transcription factors (proteins) aid attachment of RNA polymerase
- 3 types of RNA polymerase
- TATA box -- in promoter region; helps identify where RNA polymerase should bind
- More transcription factors attach after RNA polymerase attaches
- RNA polymerase unwinds DNA at start point of gene
- 2) Elongation
- RNA polymerase unwinds DNA and base pairs RNA nucleotides to DNA gene
- RNA is made 5’ to 3’ so DNA gene is 3’ to 5’
- Proceeds until RNA polymerase encounters sequence that triggers its dissociation
- In eukaryotes, RNA polymerase actually passes termination point before RNA molecule is released
- 3) Termination
- Intrinsic termination -- transcription stops when newly synthesized RNA molecule forms a G-C (bond) rich hairpin loop followed by a run of U’s; when loop forms, mechanical stress breaks down weak rU-dA bonds; pulls poly U-transcript out of active site of RNA polymerase, terminating transcription
- Extrinsic termination -- protein factor called rho destabilizes interaction between template and mRNA, releasing newly synthesized mRNA from elongation complex
- 4) RNA processing
- ONLY IN EUKARYOTES
- 5’ cap w/ modified guanine nucleotide is added
- Poly-A-tail is added
- At 3’ end; consists of 30 to 200 adenine nucleotides
- Prevents mRNA from being degraded
- Signal ribosome where to attach
- Determines how many times mRNA can be translated before it’s destroyed
- Average immature RNA is 8000 nucleotides but mature mRNA is 1200 nucleotides
- Introns -- non coding regions that REMOVED in eukaryotic cells
- Exons -- remaining regions; called this because their genes are EXpressed; joined together to form cistron
- Removing Introns
- Spliceosome removes introns
- Spliceosomes = smaller particles called snRNP (made of proteins and snRNA)
- Spliceosome will splice the intron at a specific RNA sequence releasing a “lariat” RNA
- RNA processing -- different exons are recombined in different ways for certain mRNAs; increases the number of different proteins
- Exon shuffling and different proteins
- Proteins have modular architecture consisting of discrete regions called domains
- Different exons code for different domains in protein
- Exon shuffling may result in evolution of new proteins
DIFFERENCES IN PROTEIN SYNTHESIS BETWEEN PROKARYOTES AND EUKARYOTES
Prokaryotes
| Eukaryotes
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Protein Synthesis Translation
- Involves mRNA → protein
- Materials:
● Ribosome
● tRNA w/ correct amino acid connected
1. tRNA= transfer RNA
2. Job: helps decode mRNA sequence into protein
● Mature mRNA
1. mRNA= messenger RNA
2. Job: carries genetic code from DNA to ribosomes (single-stranded molecule)
- Ribosome Structure:
● Made from proteins + rRNA
● 2 subunits *only together when ribosome = translating
1. Small subunit
2. Large subunit
+ Polyribosomes translate same piece of mRNA
● Three sites where tRNA attaches
1. A site= tRNA arrives w/ amino acid
2. P site= has a tRNA that attaches to tRNA at A site
3. E site= tRNA exits w/o amino acid
- tRNA Structure + Info
● Folded structure with 3 hairpin loops
● tRNA brings correct amino acid to mRNA
● Self vase pairing allows tRNA to form clover leaf shape
● At end of tRNA there are 3 nucleotides, base pair with mRNA codons using complementary pairing AKA anticodon
EX: mRNA= AUG THEN tRNA= UAC
● Amino acid attached to 3’ end at ACC position
● Should be 61 deff types of tRNA because there are 61 anticodons HOWEVER because of redundancy at position #3 on mRNA codon base pairing rule = not as strict at position #3 as first 2 RESULT= ONLY 45 tRNA molecules
- 3 of codons are stop codons → 64-3= 61 codons
- Relaxing of pairing between third base of codon + anticodon = wobble
1. Wobble position: U on anticodon can bind w/ A OR G in 3rd position of codon
● EX: wobble effect: UCA AND UCG code for serine, if tRNA anticodon is AGU AND U will pair w/ A OR G, tRNA will still bring amino acid serine
2. Some tRNA anticodons include a modified form of adenine, inosine, which can hydro bond with U,C, or A on codon
- Attaching an Amino Acid to a tRNA
● Gets amino acid by combo w/ charging enzyme (aminoacyl-tRNA-synthetase= ATS)
+ ATS enzyme naturally binds correct amino acid on to tRNA according to anticodon
+ ATP NEEDED FOR THIS PROCESS
- Initiation of Translation
● Parts
1. Initiation→ small subunit attach to mRNA at AUG (start), tRNA w/ anticodon UAC attach to P site, then large subunit attach to small subunit
2. Elongation → 2nd tRNA arrives + attaches at A site w/ correct anticodon + correct amino acid, peptide bond formed between amino acids + water removed, then mRNA moves down one codon (AKA TRANSLOCATION), tRNA at P site relocated and releases amino acid stays on growing polypep chain, tRNA moves from P to E Site, tRNA at A site relocated to P site, new tRNA arrive starting cycle all over again, the will happen multiple times UNTIL ↡
3. Termination → at stop codon (stop protein → release factor) attach to A site, cause release of last tRNA on polypep chain, cause ribosome to fall apart
- Exported Proteins
● EP tagged + interact w/ signal recognition particles (SRP)
● SRP thread synthesized protein into lumen of ER (endoplasmic reticulum)
● Proteins exported have signal tag → interact w/ SRP which it recognizes + attaches to
● SRP attracted to protein pore on ER
● Synthesized protein threaded into cisternae of eR
● Ribosome detaches once protein made
- AMINO ACIDS AGAIN?!
● 20 Aminos (some say 21 or 22 too)
● R represent side-chain specific to each amino
Protein Structure
- Base Pair Mutation and Effect on Proteins
● 3 types of mutations
1. No effect on protein structure (because of redundancy of universal code)
2. Missense mutation→ change in one amino (EX: intended: GGC (glycine) → result: AGC (serine)
3. Nonsense mutation→ shortening of polypep chain (EX: intended: AAG (lysine) → result: UAG (stop)
● Point mutation diseases
1. Cystic fibrosis caused by mutation in gene of cystic fibrosis transmembrane gene, most common mutation = deletion of 3 nucleotides that results in loss of amino acid phenylalanine (F) at 508th position on protein
2. Sickle cell anemia involves point mutation, hemoglobin chain glutamic acid is substituted w/ valine, makes chain slightly less soluble in cytosol
+ ADDITIONAL INFO
1. Result of base pair insertion/deletion substitution result in frameshift mutation leads to missense protein
2. Immediate stop codon result in immediate nonsense
3. Three missing nucleotides means 1 amino missing + no frameshift has been caused
4. Frameshift mutation EX: tay-sachs disease→ usually 4 base pair insertion in HEXA gene (chromosome 15) renders premature stop codon leads to deficiency of one of subunits of hexosaminidase protein
5. NOT ALL FRAMESHIFT AND POINT MUTATIONS RESULT IN UNSTABLE, INACTIVE PROTEINS → they can lead to genetic adaptation because of DNA change, and leads to species evolution
POSSIBLE ESSAY QUESTIONS FROM POWERPOINTS
1) Explain the role of RNA polymerase and spliceosomes (snRNPs) in protein synthesis.
a) RNA polymerase is an enzyme that unwinds DNA and uses nucleotides to assemble a mRNA strand during transcription. In eukaryotes, RNA polymerase attaches to the promoter region of the DNA, with the help of transcription factors. It then grabs free nucleotides and uses them to form an mRNA strand. It detaches off the DNA strand through extrinsic or intrinsic termination when it reaches a signal telling it to do so. Extrinsic termination occurs when the transcription factor rho signals it to stop, while intrinsic termination occurs with the creation of a G-C bond rich hairpin loop. Meanwhile, spliceosomes help with processing the mRNA that is made from transcription. Spliceosomes will excise the introns in an immature mRNA and join the exons together. The introns are then converted to free nucleotides that can be reused in protein synthesis.
2) Explain the organization of the genetic material in prokaryotes and eukaryotes and contrast the process of transcription in prokaryotes and eukaryotes.
a) The genetic material in prokaryotes is circular, while in eukaryotes it is linear. The genetic material in prokaryotes floats around the cytoplasm, while in eukaryotes it is contained in the nucleus. The process of transcription in prokaryotes and eukaryotes differs because in prokaryotes, RNA does not need to be processed, while eukaryotic mRNA needs the guanine cap and poly-A-tail. RNA in prokaryotes also has no introns while it does in eukaryotes. Transcription and translation can occur simultaneously in prokaryotes, while transcription comes before translation in eukaryotes.
3) Explain role of codons, ribosomes, and tRNA in protein synthesis in eukaryotic cells.
a) Codons are nucleotide sequences that consist of three nucleotides each coding for a unique one amino acid, there are about 20-22 of them. Ribosomes are known as the site for protein synthesis, they manufacture of protein takes place in this organelle. tRNA helps match specific amino acids to corresponding codons on mRNA for polypeptide chains.
4) Explain how a single base-pair mutant in DNA can alter the structure and function of a protein. Using a real life example discuss the potential consequences of the production of a mutant protein to the structure and function of the cells of an organism.
a) A single base-pair mutant occurs when one nucleotide is accidentally added or deleted. This results in a frameshift mutation that changes the amino acid that is being coded. This frameshift mutation has the potential to make a small error or a life threatening mistake. If the frameshift mutation makes a new codon that codes for the same amino acid as the old one, then the error will often be overlooked. But if the frame shift mutation codes for an entirely new amino acid, then the structure and function of the protein will be damaged. For example, in Tay-Sachs disease, the frame shift mutation causes a premature stop codon which damages hexosaminidase. This enzyme breaks down lipids in the brain, but the frame shift mutation changes its shape and causes it not to function properly.
5) How is translation different in prokaryotes and eukaryotes?
a) In prokaryotes, translation can occur simultaneously with transcription. In eukaryotes, translation comes after transcription. Prokaryotes cannot export proteins due to the lack of an endoplasmic reticulum. They will not have the signal recognition particle (SRP) that eukaryotes have. In eukaryotes, SRP helps move the newly synthesized protein to the ER so it can go out of the cell.
Chapter 14 Review:
Gene specify proteins via transcription + translation
- Beadle + Tatum studies of Neuropora led to 1 gene-one polypep hypothesis: during gene expression, info coded in genes makes specific polypep chains, enzymes, other proteins or RNA molecules
- Transcription: synthesis of RNA complementary to a template strand of DNA.
- Translation: synthesis of polypep whose amino acid sequence is specified by nucleotide sequence in mRNA (Messenger RNA)
- Codons: nucleotide triplet
- Codon in mRNA translated into amino acid (61 of 64 codons)/ serves as stop signal (3 codons)
- Codons MUST BE READ IN CORRECT READING FRAME
Transcription is the DNA-direction synthesis of RNA
- RNA polymerase (links together RNA nucleo. Complementary to DNA temp strand) catalyzes RNA synthesis
- 3 stages of transcription= initiation, elongation, termination
- Promoter (establishes where RNA synthesis is initiated) includes TATA box (crucial DNA sequence) in eukaryotes
- Transcription factors help eukaryotic RNA polymerase recognize promoter sequences forming transcription initiation complex
- TERMINATION DIFFERS IN BACTERIA AND EUKARYOTES
Eukaryotic cells modify RNA after transcription
- Euk pre-mRNAs undergo RNA processing includes RNA splicing (add a modified nucleotide 5’ cap aka methyl cap and poly-A tail to 3’ end, removes introns and keeps exons)
- PROCESSED mRNA INCLUDES UNTRANSLATED REGION = 5’ UTR or 3 UTR at each end of coding segment
- Exons are joined
- RNA splicing carried out by spliceosomes but sometimes RNA alone catalyzes own splicing
- Properties of RNA allow SOME RNAs (ribozymes) to act as catalysts ex: introns splicing themselves out
- -presence of introns allows for alternative RNA splicing (difference is that RNA splicing is process of splicing exons of primary transcript of mRNA but alt splicing is process of making different combos of exons of same gene so some exons are exons are skipped resulting in various forms of mature mRNA)
Translation is the RNA-directed synthesis of a polypeptide
- Cell translated an mRNA message into protein using tRNAs (transfer RNAs)
- Amino acid bound by aminoacyl-tRNA synthetase
- tRNA lines up via anticodon at complementary codon on mRNA
- Ribosome made up of ribosomal RNAs (rRNAs) and proteins facilitates coupling w/ binding sites for mRNA and tRNA
- Translation has 3 stages: initiation (A site), elongation (P site), termination (E site), remember APE and that A= Amino acid enter attach to tRNA w anticodon P= polypeptides formed here E= exit of tRNA
- After translation modification to proteins affect their shape
- Free ribosomes in cytosol initiate synthesis of all proteins but proteins w/ signal peptide synthesized on ER
- Gene can be transcribed by mult. RNA polymerases simultaneously
- Single mRNA can translated simultaneously by number of ribosomes forming polyribosome
- In bacteria, processes are coupled
- However in euk they are separated in time and space by nuclear membrane
Mutations of one or a few nucleotides can affect protein structure and function
- Point mutations: changes in one DNA nucleo pair, lead to production of non-func proteins
- Nucleotide-pair substitutions: cause missense/nonsense mutations
- Nucleotide-pair insertions/deletions may produce frameshift mutations
- Spontaneous mutations can occur during DNA replication, recombination, repair
- Chem and physical mutagens (x-rays, gamma rays, alpha particles BASICALLY IONIZING RADIATIONS) cause DNA damage that can alter genes
Protein Synthesis Study Guide
● Review the general information about proteins - the shape, structure and composition
● Review the amino acids and their chemical makeup as well as the different R groups and the effect they have on the protein's shape.
● Know the different RNA's used in transcription/translation and what their jobs are.
● Know how RNA is made - what direction, what proteins are used, where it happens
● Review RNA processing - what happens, what does it, and why
● Know the different kinds of mutations - what the are, what their effect on the protein is and which ones are worse
● Know what causes mutations
● Understand why/how a mutation effects proteins and their function
● Understand the difference of the effect of a mutation in gametes vs an single cell of a multicellular organism
● Be able to transcribe mRNA from DNA and translate mRNA into amino acids
● Know the steps of what happens at the ribosome during translation
● Be able to compare and contrast transcription and translation in prokaryotes
● Math
o n^r
o math based on codons
https://secure-media.collegeboard.org/apc/ap09_biology_q4.pdf
a) Role of each?
1. RNA polymerase → DNA is a template to make the mRNA
2. Spliceosomes → removes introns and connects the exons in RNA
3. Codons → code for amino acids
4. Ribosomes → mRNA attaches to here as it is the site of protein synthesis and it helps make the protein
5. RNA → transports amino acids along with anticodons
b) Mechanism + Job
1. RNA editing → removing of introns connects the exons together
2. Promotor → increases RNA polymerase binding
3. RNA processing → adds the GTP cap or Poly-A tail w/ 5’ / cap
c) Virus + explanation
1. HIV → RNA virus
2. The central dogma is the unidirectional flow of genetic information from DNA to mRNA but in HIV there is central dogma reverse so the flow of genetic information is in the reverse direction. An enzyme helps synthesize DNA from genetic RNA. So it goes from RNA to DNA.
https://secure-media.collegeboard.org/apc/ap12_biology_q3.pdf
a) Describe role of: RNA splicing, repressor proteins, siRNA
1. RNA splicing- exons spliced together, introns removed, spliceosomes help remove introns
2. Repressor proteins- inhibit transcription and inhibit translation, silence genes, inactivate gene expression
3. siRNA- facilitates degradation of mRNA and inhibits translation
b) Mutations
1. Silent- nucleotide change- no change in amino acid/protein sequence
2. Missense substitution- nucleotide change causes new codon- different amino acid/protein sequence
3. Nonsense substitution- nucleotide change causes stop codon- protein not formed
c) Environmental factors
1. UV light
2. Carcinogens- from cigarette smoke
3. WHAT IT COULD DO- DNA altered/damaged and it disrupt gene sequence
d) dna/histone modification
Other Random Bits:
● BECAUSE the gene code is nearly universal, it was there in very early life and therefore present in the common ancestor of all present-day organisms.
● The promoter region is a DNA sequence
○ Promoter includes a start point and a few dozen nucleotides upstream
● In Eukaryotes, RNA polymerase II is used for pre-mRNA synthesis
● TATA box is ONLY in eukaryotes (typically included in euk promoter)
○ TATA box is about 25 nucleotides upstream from the start point
● Promoter includes a start point
○ Start point = nucleotide where RNA polymerase starts making mRNA
● Termination
○ Prokaryotes/Bacteria:
■ DNA termination sequence is transcribed into transcribed terminator (termination signal)
■ Polymerase detaches and releases transcript
○ Eukaryotes:
■ RNA polymerase II transcribes DNA polyadenylation signal (termination signal - AAUAAA) in pre-mRNA
■ When this appears, the transcribed termination signal immediately binds to proteins in the nucleus
■ At a point some nucleotides downstream from this signal, the proteins cut the transcript from polymerase, releasing pre-mRNA
● UTRs = Parts of mRNA that won’t be translated into protein, but have other functions (ex: ribosome binding)
○ 5’ UTR
○ 3’ UTR
● If something transcribed is noncoding, then it won’t be translate (introns)
● Introns - the sequence of DNA nucleotides that code for euk. Polypeptides isn’t usually continuous
● Ribozyme = RNA mol. that functions as enzyme (rRNA)
● Snurps recognize the sequence that signal the start and ends of introns and cut them out
● A site = reads open condon and brings in new tRNA
● RNA poly I synthesizes rRNA
● rRNA also acts as a catalyst for peptide bond formation
● Ribosome positions new AAs to be added to the CARBOXYL end of the polypeptide
● Initiation factors (proteins) bring all components of translation together including ribosome subunits, mRNA, tRNA, etc.