Unit 6: Protein Synthesis and Gene Expression/Regulation Flashcards
RNA
Ribose sugar. Uracil instead of thymine. Single stranded. The sequence of the RNA bases, together with the structure of the RNA molecule determines RNA function
mRNA
carries genetic information from DNA to the ribosomes. Information is used to direct protein synthesis.
codon
three base sequence found on mRNA.
tRNA
helps create a specific polypeptide sequence at the ribosomes as directed by mRNA. Various types of tRNA, each carrying a specific amino acid
anticodon
three base sequence found on tRNA. Correct base pairing of tRNA anticodons with mRNA will result in the release and addition of an amino acid to a growing polypeptide
rRNA
functional units of ribosomes responsible for protein assembly. Base pairings of anticodons and codons occurs in the ribosome. Creates primary polypeptides as tRNA releases amino acids.
microRNA
small RNA molecules that bind to other RNA molecules to degrade them
Gene Expression
- process of using a gene to generate either a protein or a functional RNA
- The gene contains a series of nucleotides which are ‘read’ by cellular enzymes to produce a protein or functional RNA
- The order of nucleotides in the gene determines the amino acids which will be joined to produce a protein
- Different genes have different nucleotides which code for different proteins.
Where are proteins assembled in eukaryotes?
Proteins are assembled at the ribosomes, which can be free-floating in the cytoplasm or attached to the rough endoplasmic reticulum (RER) in eukaryotic cells.
Exons
- the coding regions of a gene that are retained in the mRNA and will be translated into protein
- only in eukaryotes
Introns
- the non-coding regions of a gene that are removed during RNA splicing and do not contribute to the final mRNA product or protein
- only in eukaryotes
Promoters
- region of the gene that is a specific sequence of DNA that signals the start of transcription
- usually extends several dozen nucleotide pairs upstream of the start point
TATA box
crucial promoter in forming the initiation complex in eukaryotes
RNA Polymerase and its Role in Creating mRNA
- binds to the promoter region of the DNA
- RNA polymerase reads the DNA template strand in the 3’ to 5’ direction
- It then starts synthesizing an mRNA strand by adding complementary RNA nucleotides (A, U, C, G) in the 5’ to 3’ direction.
- When RNA polymerase reaches the terminator sequence of base pairs on the DNA template strand, it completes the production of pre-mRNA and releases it into the nucleoplasm.
base pairs in RNA
A (adenine) pairs with U (uracil)
G (guanine) pairs with C (cytosine)
transcription factors
helps RNA polymerase bind to the promoter
In eukaryotes RNA polymerase joins with several transcription factor proteins at the promoter (a special sequence of base pairs on DNA that signals the beginning of a gene)
This combination is called the transcription initiation complex.
template strand
- The DNA strand that RNA polymerase adds bases to in 5’ to 3’ direction
- also called the noncoding strand, minus strand, or antisense strand
- mRNA strands base pairs are going to be complementary to the base pairs on this
coding strand
- the side of the DNA that is not used by RNA polymerase because it is not in the 3’ to 5’ direction
- the base pairs for the mRNA are the same as the base pairs for the coding strand except with U switched with T because it was coded to be complimentary with the template strand
terminators
- region of the gene that tells the RNA polymerase to stop coding the strand
- in prokarytes, the mRNA will go straight to being translated after this
- in eukaryotes, the mRNA has to be processed, so it is just pre-mRNA now
pre-mRNA
- initial form of mRNA that is transcribed from DNA in the nucleus of eukaryotic cells before it undergoes processing to become mature mRNA
- It contains both introns (non-coding regions) and exons (coding regions)
- eukaryotes only
Processing of Pre-mRNA: what is added on
- A poly-A tail is added to the 3’ end of the mRNA transcript
- This modification helps protect the mRNA from degradation, aids in the export of the mRNA from the nucleus to the cytoplasm, it protects it rom hydrolytic enzymes, and plays a role in the initiation of translation
- A GTP cap is added to the 5’ end of the mRNA transcript, it is a modified guanine nucleotide
- It helps protect the mRNA from degradation, facilitates the binding of the ribosome for translation, and aids in the export of the mRNA from the nucleus
- this is done by enzymes in the nucleus
Processing of Pre-mRNA: splicing
- Splicing done by spliceosomes
- stay in the nucleus; do not code for proteins. They are non-coding sequences (introns are in between sequences)
- exit the nucleus to go to the ribosome; do code for proteins
- They are the real genes
- They code for proteins (exons expressed)
Alternative splicing
Alternative mRNAs produced from the same gene
Translation: Initiation
- A cell translates an mRNA message into protein with the help of transfer RNA (tRNA)
- Small ribosomal subunit binds to mRNA and an initiator tRNA, Then the large ribosomal subunit attaches
- Each tRNA molecule carries a specific amino acid to the ribosome based on the mRNA’s codons
Translation: Elongation
- The ribosome moves down the mRNA in the 5’ to 3’ direction
- For each codon (3 bases on the mRNA), a tRNA with a corresponding anticodon brings an amino acid to the ribosome
- The amino acid is added to the preceding one by a peptide bond
- If the anticodon on the tRNA is complementary to the codon on the mRNA → the correct amino acid is at the ribosome and gets added
rRNA’s role in elongation
- During translation, rRNA helps catalyze the formation of peptide bonds between amino acids, allowing the protein chain to elongate
- Essentially, rRNA provides the structural and catalytic functions required for protein synthesis to occur efficiently within the ribosome
- also helps align the mRNA and tRNA correctly within the ribosome to ensure the accurate synthesis of proteins
the “wobble”
how multiple base pairs in a codon can code for the same amino acid during elongation
Translation: Termination
- Elongation continues until the ribosome reaches a stop codon in the mRNA, UAG, UAA, or UGA, A protein called a release factor that causes the polypeptide chain to separate from the ribosome
- The polypeptide folds up based on the arrangement of its amino acids (secondary and tertiary protein structure)
- Some polypeptides combine with others to make larger proteins (quaternary structure)
- May be packaged at ER or modified and packaged at the Golgi
Protein Synthesis in Prokaryotes vs. Eukaryotes
Prokaryotes:
- Transcription occurs in the cytoplasm
- No mRNA editing/processing
- Transcription and translation occur simultaneously
- no introns
Eukaryotes:
- Transcription occurs in the nucleus
mRNA is edited prior translation
- Translation occurs after transcription is completed
- has transcription factors, exons, introns, and pre-mRNA
Substitution mutation
A mutation where one nucleotide is replaced by another in the DNA sequence.
Missense mutation
A substitution mutation that results in a different amino acid being incorporated into the protein, potentially altering its function
Silent mutation
A substitution mutation that does not change the amino acid sequence of the protein, often due to the wobble effect
Nonsense mutation
A substitution mutation that converts a codon into a stop codon, leading to premature termination
Insertion
A mutation where one or more nucleotides are added into the DNA sequence
Deletion
A mutation where one or more nucleotides are removed from the DNA sequence.
Frameshift Mutation
A mutation caused by insertions or deletions that shift the reading frame of the genetic code, often altering the entire amino acid sequence
Operon
- entire stretch of DNA that includes the operator, the promoter, and the genes that they control
- only in prokaryotes
Operator
regulatory sequence that controls access to the genes by allowing or blocking RNA polymerase based on the presence of repressors or activators
Repressor Molecule
- The operon can be switched off by a protein repressor
- The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase
- The repressor is the product of a separate regulatory gene
- A repressor protein can be in an active or inactive form, depending on the presence of other molecules
- almost always a protein
Co-repressor
A corepressor is a molecule that cooperates with a repressor protein to switch an operon off.
Inducer
An inducer helps to switch an operon on.
What happens when an inducer molecule binds to the repressor molecule?
When an inducer molecule binds to the repressor protein, the repressor undergoes a conformational change, causing it to detach from the operator, allowing RNA polymerase to transcribe the genes in the operon.
Repressible Operon
- from on to off
- Usually functions in anabolic [bonds are being synthesized between substrates, requires input of energy (endergonic)] pathways
- When the end product is present, transcription is repressed/turned off to allocate resources to other uses
- only in prokaryotes
Trp operon
- repressible operon
- The group of genes in this operon help the organism to produce the amino acid tryptophan from other compounds when tryptophan is not present in the cell’s environment
- When there is low tryptophan present, Trp repressor stops binding to the operator so transcription can happen and tryptophan can be made
- When there is large amounts of tryptophan present, Trp repressor becomes active from the corepressor molecule
- The repressor is active only in the presence of its corepressor tryptophan - thus the trp operon is turned off (repressed) if tryptophan levels are high
- only in prokaryotes
Inducible operon
- from off to on
- Usually functions in catabolic [bonds are being hydrolyzed, releases energy (exergonic)] pathways. Produces enzymes only when the nutrients are available (avoids making proteins that have nothing to do)
- Turned off when nutrients are NOT available
- only in prokaryotes
Lac Operon
- inducible operon
- Contains genes that code for enzymes used in the hydrolysis and metabolism of lactose
- If lactose is present, it acts as an inducer that binds to inactivate the repressor
- Turns the operator on since need to make enzymes to digest lactose (turn on genes)
- only in prokaryotes
Stem cells
- undifferentiated cells that have the potential to become other cell types
- eukaryotic
Differentiated Cells
Cells turn on the parts of DNA they need and off those they don’t → cell differentiation through regulatory proteins
Different Ways to Regulate Which Genes a Cell Expresses
- Regulate chromatin structure
- Regulate transcription initiation
- Post-transcriptional regulation
Epigenetic changes
- do not change your DNA sequence, but they can change how your body reads a DNA sequence
- only in eukaryotes
Eukaryotic Gene Expression: Regulating Chromatin Structure
- DNA is wrapped around proteins called histones
- Histone acetylation - acetyl groups are added to histones, which prevents them from binding the DNA as tightly, making room for proteins to bind for transcription
- DNA methylation - methyl groups can attach to DNA bases which makes the DNA physically tighter and more compact, preventing transcription
- both are examples of epigenetic changes
- When chromatin is loosely packed (euchromatin), the DNA is more accessible to transcription factors and RNA polymerase, so the gene is more likely to be transcribed
- When chromatin is tightly packed (heterochromatin), the DNA is less accessible, and gene expression is typically repressed
Eukaryotic Gene Expression: Regulating Transcription Initiation
- Transcription factors are proteins that bind upstream of the gene
- Activators help RNA polymerase bind to the DNA, increasing transcription
- Repressors prevent RNA polymerase from binding to the DNA, preventing transcription
Post-transcriptional Regulation
- RNA splicing: different mRNA molecules are produced from the same primary transcript depending on which exons are used
- mRNA degradation: nuclease enzymes break down mRNA; lifespan of mRNA varies
- Initiation of translation: if the ribosome does not form or mRNA cannot attach, translation does not occur
- Protein processing and degradation: many proteins need to be modified to become active; may be modified by the addition of ubiquitin, which triggers proteasomes to break down the protein
- microRNAs can bind to mRNA, causing it to degrade or blocking it from being translated
Differences Between Prokaryotic and Eukaryotic Gene Expression
Prokaryotes:
- Clusters of genes (operon) regulated
- Regulation only occurs in the cytoplasm
- No histones
- No introns (every part codes for the protein)
- Regulation only at transcriptional level (operons)
- Why? Because transcription & translation are simultaneous; there’s not a chance to regulate once transcription starts
Eukaryotes:
- Individual genes regulated
- Regulation occurs in the nucleus and cytoplasm
- DNA wrapped around histones (allows for epigenetic regulation)
- Introns → (alternative) splicing
- Regulation at epigenetic level, transcriptional level, post-transcriptional level, translational level, and post-translational level
Restriction Enzymes
- Also known as restriction endonucleases
- Cut DNA into segments at specific sequences
- Can be used for gel electrophoresis and bacterial transformation
- Leave behind “sticky ends”
Biotechnology: Gel Electrophoresis
- Electricity is used to separate
- DNA fragments of different sizes
- DNA is negatively charged due to phosphate groups
- Moves towards the positive end of the electrophoresis chamber
- Smaller molecules (with lower molecular weight) will travel farther
Biotechnology: Bacterial Transformation
- Allowing organisms to express genes (synthesize proteins) that originate from other organisms
- Uses plasmids - small self-replicating circular DNA molecules
- Genetically engineered (recombinant) plasmids are inserted into bacterial cells
- The recombinant plasmid contains additional genes from another source (“gene of interest”)
- Restriction endonucleases are utilized to create cleavage sites, forming sticky ends that join to form a recombinant molecule
- When inserted into bacteria, the bacteria will express the new genes
- Bacteria are opportunists that will take up DNA from their environment
- Ex: bacteria producing insulin, insecticide resistant corn, glow-in-the-dark fish
Biotechnology: DNA Sequencing
- Goal is to determine sequence of bases in a sample of DNA
- Fluorescent markers are added to PCR
- The fragments are then separated by size
- The order of colors from the markers are recorded; each color representing a different base
Biotechnology: Polymerase Chain Reaction (PCR)
- Process of amplifying (making many copies of) DNA for analysis
Steps: - Denaturation - heat briefly separates the DNA strands (Polymerase requires an open 3’ end)
- Annealing - cool to allow primers to form hydrogen bonds with ends of target sequence
- Extension - Taq Polymerase (a type of DNA polymerase that can withstand the heat) add nucleotides to the 3’ end of each primer
Biotechnology: CRISPR
- Clustered Regularly Interspaced Short Palindromic Repeats
- Used naturally in bacteria to repair against viral damage
- Gene editing technology that may allow curing of genetic diseases
- Bacteria use CRISPR to remember viruses that infected them before.
- They save bits of viral DNA in their own genome as “spacers” between repeating sequences.
- If the virus comes back, the bacteria use those saved sequences to recognize the virus and cut up its DNA
- Cas9 → The enzyme that acts like molecular scissors
- Guide RNA (gRNA) → Tells Cas9 where to cut by matching the DNA sequence
- Target DNA → The specific DNA sequence that scientists want to cut or edit
Biotechnology: Cloning
Using the nucleus from a somatic (body) cell from one organism and an enucleated egg cell from another organism
Retroviruses
- Retroviruses have an alternate flow of information: from RNA to DNA
- Made possible by reverse transcriptase, an enzyme that copies the viral RNA genome into DNA
- This DNA integrates into the host genome and becomes transcribed and translated for the assembly of new viral progeny
- Have a single-stranded RNA genome, a protein coat (capsid), and an outer lipid envelope.
- Once integrated, the viral DNA is transcribed and translated by the host cell to produce viral RNA and proteins, which are assembled into new virus particles.
- An example of this is HIV which turns into AIDS
what bond is formed between the amino acids during protein synthesis?
remember amino acids are:
H
|
amino group - C - carboxyl
|
R group
so the carboxyl group of the first amino acid forms a peptide bond (covalent bond between two amino acids) with the amino group of the next one
