Chapter 17 - From Gene to Protein Flashcards
Gene Expression
the process by which DNA directs the synthesis of proteins by transcribing a mRNA sequence and translating it to produce a polypeptide chain
Differences in Transcription and Translation between Eukaryotes and Prokaryotes
- Bacteria do not have nuclei therefore there is no membrane separating DNA and mRNA from ribosomes
- Allows mRNA synthesis to begin while it is being transcribed
- Eukaryotic cells have an envelope separating the two processes over space and time
- Eukaryotic cells also create a primary transcript which has to undergo modifications to produce final functional RNA transcript
Genetic Code
the genetic instructions for a polypeptide chain are written in the DNA as a series of nonoverlapping three nucleotide words
- These are translated into 3 nonoverlapping words in mRNA before translated into an amino acid
Important Features of the genetic Code
- Two codons can code for 1 amino acid = redundancy
○ Codons often differ only in the third nucleotide base- 1 codon cannot code for 2 amino acids = no ambiguity
○ Each codon codes for a specific amino acid
- 1 codon cannot code for 2 amino acids = no ambiguity
Components of Transcription
RNA polymerase pries the two strands of DNA apart and joins together RNA nucleotides in a 5’-3’ direction
○ Don’t need a primer and can start from scratch
Promotor: specific sequences of nucleotides which mark where transcription of a gene begins and ends and where the RNA polymerase attaches to
Transcription Unit: the stretch of DNA downstream from the promotor that is transcribed into the RNA molecule
Terminator: sequence which signals the end of transcription
Transcription Initiation Complex
the complete assembly of transcription factors and RNA polymerase bound to a promotor
- TATA box is the sequence of DNA which helps form
Stages of Transcription
- Initiation:
- RNA polymerase binds in a specific orientation on the promotor after transcription factors are attached allowing it to unwind the DNA strands
○ Transcription factors in eukaryotes guide the binding of polymerase and initiation of transcription.
- Elongation:
- As polymerase moves downstream along the DNA, it untwists the helix exposing 10-20 nucleotides at a time and adds complementary nucleotides to the 3’ end of the growing RNA molecule
- The growing RNA strand peels away and the DNA strand reforms a double helix - Termination:
- In bacteria, transcription goes through a terminator sequence, causing the polymerase to detach and release the RNA transcript which then undergoes further modifications
- In eukaryotes, RNA polymerase 2 transcribes a polyadenylation signal sequence which is a stretch of 6 RNA nucleotides
- This results in enzymatic proteins cutting the RNA transcript free from the polymerase about 10-35 nucleotides further downstream, releasing pre-mRNA.
- The enzymes then catch up to the polymerase and it dissociates from DNA
- RNA polymerase binds in a specific orientation on the promotor after transcription factors are attached allowing it to unwind the DNA strands
RNA processing
the modification of RNA primary transcripts including splicing of introns, joining of exons and alterations of 5’ and 3’ ends to produce a mature mRNA
- 5' receives a 5' cap: a modified guanine nucleotide, after the initiation of transcription - 3' receives a poly A tail: sequence of 50-250 adenine nucleotides, at the termination of transcription
Functions of end RNA processing modifications
- Cap:
○ Facilitate the export of mature mRNA out of the nucleus
○ Help ribosomes attach to the 5’ end of the mRNA molecule- Poly A Tail:
Help protect the mRNA from degradation by hydrolytic enzymes due to mRNA’s temporary nature
- Poly A Tail:
RNA splicing
the removal of introns in DNA and the joining together of exons
- Introns: noncoding segments
- Exons: coding segments
- Carried out by a spliceosome
Importance of exons
- A single gene can encode for more than one kind of polypeptide based on which segments are treated as exons during RNA processing
Alternative RNA splicing: different mRNA molecules are produced from the same primary transcript due to different segments being treated as introns and extrons
Domains: a discrete structural and functional region of a protein
2. Different exons can code for different domains of a protein, creating different active sites and catalyzing different reactions
3. Introns also facilitate the evolution of new proteins because of exon shuffling where introns increase the probability of crossing over between the exons of alleles Creates no combinations of exons and therefore proteins with different structures and functions
Transfer RNA
transfers an amino acid from the cytoplasmic pool to a growing polypeptide chain
- bear a specific amino acid at one end of its 3d shape, while at the other end is a nucleotide triplet: anticodon,
Structure of tRNA
Consists of a single RNA strand which has both 5’ and 3’ ends folded and located near one end of the structure
○ 3’ end is the attachment site for each amino acid
○ The loop on the opposite side of the structure contains the anticodon
Aminoacyl-tRNA synthetase:
an enzyme joining the correct amino acid to tRNA
○ There are 20 different Aminoacyl-tRNA synthetases one for each amino acid, which binds that amino acid to an appropriate tRNA
○ Catalyzes the covalent attachment of the amino acid in a process driven by ATP hydrolysis resulting in a charged tRNA
Wobble
flexibility in base pairing rules in which the nucleotide at the 5’ end of a tRNA anticodon can form hydrogen bonds with more than one kind of base pair at the 3’ end of a codon
- there are not enough types of tRNA molecules for each one to be specific to each possible codon so flexible base pairing occurs / wobble
Ribosomes
- Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis
- They are composed of a larger and smaller subunits, each made up of proteins and a ribosomal RNA
- The ribosome holds the mRNA and tRNA in close proximity so the next amino acid can be added to the carboxyl end of the growing polypeptide
- It then catalyzes the formation of the peptide bond
- Before passing the polypeptide through the exit tunnel in the larger subunit
tRNA binding sites at a ribosome
- A site: holds the tRNA molecule carrying the next amino acid
- P site: holds the tRNA carrying the growing polypeptide chain
- E Site: where discharged tRNA’s leave the ribosome
Stages of Translation: Initiation
- A small ribosomal subunit binds to the mRNA followed by the specific initiator tRNA molecule, carrying the first amino acid methionine
○ The mRNA sequence is bound at the specific RNA sequence, just upstream of the start codon
○ The initiator tRNA molecule and small subunit is bound to the 5’ cap of mRNA and scans downstream until it reaches the start codon- The larger subunit then binds to the tRNA molecule at the P site to form a Translation initiation Complex
- this is brough together by initiation factors
This leaves the tRNA in the P site, and the A site is vacant for the next tRNA molecule
Stages of Translation: Elongation
- Amino acids are added one by one to the previous amino acid at the C-Terminus end through the assistance of elongation factor proteins
1. Codon recognition requires hydrolysis of GTP to increase accuracy and efficiency
2. rRNA molecule in large subunit catalyzes peptide bond formation between growing polypeptide and amino group of new amino acid.
Another GTP is hydrolyzed to provide energy for translocation of tRNA molecules between A, P and E sites as well as provides energy to break hydrogen bonds between tRNA molecule in E site and release it.
Stages of Translation: Termination
- Elongation continues until a stop codon is read and brought to the A site, bringing release factors to the A site
- This results in the addition of a water molecule instead of an amino acid, which hydrolyzes the bond between completed polypeptide and tRNA in P site, releasing the polypeptide
- Hydrolysis of 2 more GTP molecules occur in order for the release of remaining tRNA molecules breakdown of the ribosome subunits
Post Translational Modifications
- modification of certain amino acids by the attachment of sugars, lipids, phosphate groups or other additions in order for the protein to do its job
- The removal of one or more amino acids by enzymes from the amino end of the chain
- The chain can be enzymatically cleaved into two or more pieces in order for them to be activated
- Two or more polypeptide chains synthesized separately can come together for quaternary structure.
Relationship between location of ribosome and its purpose
- The location of the ribosome determines where the synthesized polypeptide chain goes
○ Free ribosomes suspended in the cytosol synthesize proteins which function in the cytosol
○ Bound ribosomes attached to the ER make proteins for the endomembrane system
Signal Peptide
a sequence of amino acids which targets the ribosome to bind to the ER
- Polypeptide synthesis always begins in the cytosol on a free ribosome unless the growing polypeptide cues the ribosome to attach to the ER
Signal Recognition Particle
a protein RNA complex which recognizes and binds to the ribosome carrying the signal peptide as it emerges, dragging the ribosome to the ER by binding now to a receptor protein on the ER
Polyribosomes
a group of several ribosomes attached to, and translating, the same messenger RNA molecule
- allows a single mRNA is used to make many copies of a polypeptide simultaneously
Substitution
the replacement of one nucleotide and its partner with another pair of nucleotides
- Some have no effect due to the redundancy of the nuclear code(more than one codon translates for the same amino acid)
Types of substitution Mutations
- Silent Mutation: a substitution mutation with no observable effect due to the changed codon coding for the same amino acid
- Missense Mutations: a substitution mutation resulting in a codon which codes for a different amino acid
○ Can have little effect on the protein as it may have similar properties or be in a region of the protein which is not essential
○ Can be detrimental if affecting an area crucial for the protein function e.g. the active site - Nonsense mutation: a substitution mutation resulting in a codon coding for a stop amino acid, resulting in shorter and nonfunctional proteins
- Missense Mutations: a substitution mutation resulting in a codon which codes for a different amino acid
Insertions and Deletions
the addition or loss of nucleotide pairs in a sequence
Can alter the reading frame of the genetic message; frameshift mutation
Spontaneous Mutations
when an incorrect nucleotide is added to a growing chain and is mismatched with the nucleotide base on the complementary strand, resulting in an incorrect base and a faulty template strand if not fixed by DNA proofreading
Mutagen
a chemical or physical agent that interacts with DNA and can cause a mutation
