mcat bio #2 - amazon s3 · dna polymerase α synthesizes first stretch of dna from rna primers (~20...

CLUTCH MCAT / BIO #2: RNA & The Genetic Code

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MCAT BIO #2 RNA & The Genetic Code



CONCEPT: GENES TO PROTEINS ● The central dogma of molecular biology:

1. Transcription – the code of DNA is read ______________, and transcribed ______________ into an RNA code

2. RNA processing – in eukaryotes, hnRNA must be processed into an mRNA transcript before it leaves the nucleus

3. Translation – tRNAs bind to the mRNA transcript with the help of ribosomes, and polymerize amino acids N C

4. Post-translational modification – proteins undergo folding, cutting, and chemical modification

● ______________ mRNA codes for a single polypeptide, and is generally found in eukaryotes.

● ______________ mRNA codes for multiple polypeptides that tend to have related functions, usually found in prokaryotes.

● Prokaryotes can transcribe and translate genes simultaneously, because their DNA is not separated from the cytosol.



CONCEPT: TRANSCRIPTION ● Transcription has three stages: _____________________, _____________________, _____________________. ● Stage 1: ________________ – RNA polymerase associates with the template strand of DNA

□ ___________ separates the two strands of DNA, and ________________ relieves the strain on the DNA strands around the bubble, allowing RNA polymerase to bind the promoter region

□ Most eukaryotic promoters have a TATA box (~30 base pairs) upstream of the transcription start site. □ Transcription factors – proteins that help RNA polymerase bind to the promoter in eukaryotes □ σ factor subunit detects the -10 and -35 elements of promoter and helps RNA polymerase bind in prokaryotes

□ RNA pol I: synthesizes __________ in the nucleolus

□ RNA pol II: synthesizes __________ and __________ □ RNA pol III: synthesizes __________

● Stage 2: __________ - RNA polymerase catalyzes the addition of nucleotides on the growing 3’ end of the RNA transcript ● Stage 3: __________ - Poly-A signal transcribed, leading an enzyme to cut the RNA transcript downstream of the signal

● Coding strand – contains the code that will ultimately be translated, and mirrors the code found in the RNA transcript

● Template strand – contains the complementary code to the coding strand, and is read by RNA polymerase.



CONCEPT: RNA PROCESSING ● The primary transcript, or pre-mRNA, must be processed into mature mRNA before it can be translated.

● ____________ – a protective cap of modified guanine

● ____________ – a long string of adenines (100-250) added to the 3’ end of transcript that aren’t encoded by the DNA

● RNA splicing – Spliceosome excises ________________ and joins ________________

□ snRNA (small nuclear RNA) couple with snRPS (small nuclear ribonucleoproteins) to form spliceosome

□ Alternative splicing – a primary transcript may be spliced together in different ways to produce different proteins

● The poly-A tail acts like a fuse once the mRNA enters the cytosol, and ensures its eventual destruction

● The start codon, AUG, always begins the coding sequence

□ Codes for __________________ in Eukaryotes, and ___________________ in prokaryotes

● The stop codons (UAA, UAG, and UGA) end the coding sequence, but don’t actually code for any amino acids.



CONCEPT: TRANSLATION ● Translation is carried out by ribosomes and tRNA, and occurs in three stages: initiation, elongation, and termination.

● tRNA (transfer RNA) – binds to codon of RNA and matches appropriate amino acid

□ Anticodon – complementary code to codon on mRNA

● Aminoacetyl-tRNA synthetases catalyze the transfer of an amino acid to the tRNA.

● Ribosome – a structure of protein and rRNA that synthesizes polypeptides from mRNA with the help of tRNA

□ ________ site (aminoacyl) – the “acceptor site” where the charged tRNA enters

□ ________ site (peptidyl) – the “polypeptide site” where the tRNA that holds the growing polypeptide chain is held

□ ________ site – the exit site, where the uncharged tRNA exits



● Initiation:

1. The small subunit binds the charged initiator tRNA, aided by proteins called initiation factors.

2. The initiation complex binds the 5’ cap in eukaryotes, or the Shine-Dalgarno sequence in prokaryotes

3. The complex scans down the mRNA to find the start codon (5’-AUG-3’) to which the anticodon (3’-UAC-5’) binds

4. The large subunit binds to the small subunit, and the initiation factors dissociate.

● Elongation:

1. A charged tRNA with the appropriate anticodon enters the A site.

2. Peptidyl transferase forms a peptide bond between amino acid in P site and one in A site, consuming GTP for energy.

3. Translocation occurs, shifting the ribosome down one codon, and shifting the tRNAs to new sites within the ribosome.

□ Elongation factors recruit charged tRNAs and cycle GTP and GDP

● Termination:

1. A protein with a similar shape to tRNA, called a release factor, recognizes the stop codon and enters the A site.

2. Release factor causes water to be “added” to the polypeptide chain, hydrolyzing the bond with the tRNA in the P site.

3. The polypeptide and tRNAs are released, and the ribosomal subunits separate.



● Most proteins need to be folded into their proper shape, and are often assisted by chaperones.

● Some proteins are chemically modified with the addition of various chemical groups.

● Some proteins contain a signal peptide that targets the to the endoplasmic reticulum.

□ Signal peptide is translated, then recognized by a protein-RNA complex called a signal-recognition particle (SRP)

□ The polypeptide is translated into the lumen of the ER.

● Membrane proteins will be incorporated into vesicle membranes and transported to the cell membrane.

● Secreted proteins are sent to the Golgi apparatus where they may be stored, modified, and ultimately secreted in vesicles



CONCEPT: BACTERIAL REGULATION OF EXPRESSION

● Transcriptional control – regulate ability of RNA polymerase to bind to a promoter and initiate transcription

● Translational control – regulate mRNA transcript degradation and translation initiation and elongation

● Post-translational control – activate or deactivate proteins

● ______________________ – a regulatory mechanism that prevents transcription, like “turning off” a gene

□ Repressible systems – gene expression is normally happening, but can repressed

● ______________________ – a regulatory mechanism that stimulates transcription, like “turning on” a gene

□ Inducible systems – gene expression is normally stopped, but can be induced

● Operon – a cluster of genes under the control of a single promoter (Jacob-Monod Model)

□ Promoter – a region of DNA that initiates transcription

□ Operator – a segment of DNA to which transcription factors bind to regulate transcription

● lac operon – an operon that allows for the transport and metabolism of lactose

□ lacI – a gene encoding a repressor protein for the lac operon

□ lacZ, lacY, and lacA – genes encoding proteins necessary to transport lactose and metabolize it.

□ Repressor – a protein that binds to DNA and inhibits the expression of a gene, or set of genes



● Lactose acts as and inducer, allowing for transcription of the lac operon.

● Glucose is used preferentially to lactose:

□ High glucose levels low cAMP because glucose metabolism prevents cAMP formation

□ Low glucose levels high cAMP levels, lac operon turned on by CAP

□ cAMP binds to CAP (catabolite activator protein), altering its conformation, and allowing it to bind the promoter

● The trp operon is normally “turned on”, giving the bacteria a steady supply of the amino acid tryptophan.

□ Tryptophan acts as a corepressor, activating the repressor protein.

● Constitutive transcription – some genes are transcribed continuously

● Facultative transcription – some genes are only transcribed when needed

● Regulon – a group of genes or operons under the control of the same regulatory protein



CONCEPT: EUKARYOTIC REGULATION OF EXPRESSION ● Eukaryotes can regulate their gene expression through the structure of DNA.

□ ___________________ – a structure of 8 histone proteins around which DNA is wound

□ ___________________ – a loosely coiled form of DNA accessible to transcription machinery

□ ___________________ – tightly coiled form of DNA that prevents gene expression

- _________________________ – poorly expressed in all cells

- _________________________ – poorly expressed in only some cells

● Histones can be acetylated, or deacetylated, to regulate gene expression.

● DNA methylation – the addition of methyl groups to A and C in DNA silencing the associated gene



● Transcriptional regulation:

□ Promoter-proximal elements – regulatory sites near promoters, related genes use same regulatory molecules

□ Basal transcription factors – general factors needed for transcription to occur that interact with the promoter

- DNA-binding domain – binds specific sequence in promoter region

- Activation domain – binds other transcription factors

● Some transcription factors are activator and repressor proteins that bind to enhancers and silencers, respectively

● Enhancer – regulatory sequence that binds activator proteins

● Silencer – regulatory sequence that binds repressors

● Mediator proteins – act as a bridge between transcription factors and RNA polymerase

● Gene duplication – some genes are duplicated allowing for increased rates of mRNA production

● Gene amplification – increased expression of a gene via duplication or enhancers



● RNA regulation:

□ Alternative splicing – proteins interact with spliceosome to determine which sequences will be spliced together

● RNA interference – RNA held by a protein complex binds to mRNA causing its degradation, or preventing translation

□ miRNA (micro RNA) and siRNA (small interfering RNA) are noncoding RNA involved in RNA interference

● Translational control generally involves initiation, but can also involve elongation and termination.

● Post-translational control:

□ Folding – proteins must be folded, often with the assistance of chaperon proteins

□ Chemical modification – many proteins are modified with non protein substituents

□ Phosphorylation/dephosphorylation – act to regulate the activity of many proteins

□ Ubiquitin tagged proteins are marked for destruction by the proteasome



CONCEPT: DNA REPLICATION ● DNA replication has three stages: initiation, elongation, and termination.

● _________________________________ – separates the two parent strands of DNA

● _________________________________ – relieves the strain on the DNA strands around the replication bubble

● _________________________________ – bind and stabilize single-stranded DNA

● Primase – synthesizes an RNA primer at the 5’ ends

● DNA polymerase synthesizes new strand by polymerizing nucleotides, using the parent strand as a template.

Role in DNA Synthesis Prokaryotes Eukaryotes

Synthesis of DNA DNA polymerase ____

DNA polymerase ____

DNA polymerase ____

DNA polymerase ____

Removal of RNA Primers DNA polymerase ____ RNase H

Replacement of RNA w/ DNA DNA polymerase ____ DNA polymerase ____



● Synthesis of new DNA only occurs in the 5’3’ direction, the result is:

● Leading strand – the strand synthesized in a continuous manner

● Lagging strand – the strand synthesized in a discontinuous manner

□ Okazaki fragments – the segments of lagging strand

□ DNA Ligase – joins together (ligates) the Okazaki fragments with a phosphodiester bond

● DNA polymerase α synthesizes first stretch of DNA from RNA primers (~20 bases), then polymerase δ and ε take over

□ Primase is part of the PRI subunit of DNA polymerase α

● DNA polymerase ε synthesizes the leading strand and DNA polymerase δ synthesizes the lagging strand

□ DNA polymerase ε and δ are highly related

● DNA polymerase γ replicates mitochondrial DNA



● Prokaryotic replication begins at a single origin of replication.

● Eukaryotic replication requires multiple origins of replication, and involves different enzymes.



CONCEPT: PROOFREADING AND REPAIR ● DNA must be replicated and maintained with a great degree of accuracy.

● DNA polymerases, including III, ε, and δ, are capable of proofreading newly synthesized DNA.

□ If a mismatch occurs during synthesis, the enzyme recognizes and removes it before continuing.

● Mismatch repair – repair mechanism to correct base pairing mismatches after synthesis is complete

● There are many repair mechanisms that function independently of replication, because DNA is constantly damaged.

● Nucleotide excision repair – repair damage from chemicals and radiation that distort the shape of DNA

□ Particularly important for repairing UV light damage, which can result in thymine dimers



CONCEPT: TELOMERES ● During eukaryotic replication, the lagging strand ends cannot be replicated by DNA polymerase δ alone.

● Telomere – a region of repetitive nucleotide sequences that protects the ends of chromatids

□ Telomerase – an enzyme that adds DNA repeat sequences to the 3’ end of the leading strand



CONCEPT: MUTATIONS

● There are three main types of mutations: beneficial, neutral, and deleterious.

● Point mutation – a change in the DNA sequence at a single base pair

□ Substitution – the replacement of one nucleotide for another

□ Transitions – when a purine is replaced with a purine, or a pyrimidine with a pyrimidine

□ Transversions – when a purine is replaced with a pyrimidine, or vice versa

● _________________ mutations – a mutation with no observable effect on phenotype

● _________________ mutations – a mutation that changes the codon for one amino acid, to that for another

● _________________ mutations – a mutation that changes a codon for an amino acid to a stop codon



● _______________________ – the addition of nucleotides into the sequence

● _______________________ – the deletion of nucleotides from the sequence

● _______________________ mutation – a mutation that shifts the reading frame of the codons

● Deletion – loss of a chromosome fragment

● Duplication – can be cause by attachment of extra chromosome fragment

● Inversion – chromosome fragment attaches in reverse orientation

● Translocation – chromosome fragment attaches to a nonhomologous chromosome

mcat bio #2 - amazon s3 · dna polymerase α synthesizes first stretch of dna from rna primers (~20...

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