RNA & Genetic Code Flashcards
mRNA
- messanger RNA
- transcribed by template DNA strands by RNA Polymerase enzymes
- takes information from DNA to ribosomes where creation of primary protein structure occurs
- goes from 5’ to 3’
- considered a sense strand
What term can be used to describe eukaryotic mRNA?
Monocistronic – each mRNA molecule translates into only one protein per product
What term can be used to describe prokaryotic mRNA?
Polycistronic – each mRNA molecule translates into multiple proteins per product using the same promoter
tRNA
- transfer RNA (anti-codon)
- brings in amino acids and recognizes the codon on the mRNA using its anticodon (3 nucleotide sequence)
- when attached to an amino acid they are charged or activated
- always attached to 3’ end that is CCA
- pairs with appropriate codon on mRNA while in ribosome
- mature versions of this are found in the cytoplasm
- overall structure is 3’ to 5’
Aminoacyl-tRNA Synthetase
- different versions of these activate specific amino acids
- require 2 ATP to create a peptide bond during translation
- also transfers the amino acid to the 3’ end of tRNA
rRNA
- ribosomal RNA
- synthesized in nucleus
- helps catalyze formation of peptide bonds and splice out its own introns within the nucleus
- forms structural and catalytic component of ribosome
Codons
- consist of 3 bases that translate into an amino acid
- 64 total
- written in 5’ to 3’ direction
- each one represents only one amino acid but most amino acids are represented by multiple codons (=degeneracy)
- during translation mRNA codon is recognized by complementary anticodon on tRNA (orientation of this base-pairing is antiparallel)
What is the start codon?
AUG
What are the stop codons?
- UAA (U Are Annoying)
- UGA (U Go Away)
- UAG (U Are Gone)
Explain the relationship between degeneracy of the genetic code and mutations
the degeneracy of the genetic code allows for mutations in DNA that do not always result in altered protein structure or function because more than one codon can specify a single amino acid
Wobble Position
- refers to the third base in a codon
- for amino acids with multiple codons the first two bases are typically the same but the third base is variable
- protects against mutations
- mutations that occur at this position are either silent or degenerate [no effect on expression of amino acid]
Expressed Mutations
mutations that affect the primary amino acid sequence of a protein
Point Mutation
- mutation that affects one nucleotide of a codon
- one DNA base is replaced with another leading to a change in one RNA nucleotide and ultimately a change in one amino acid
Frameshift Mutation
- occurs when some number of nucleotides are added or deleted from the mRNA which results in changes to AA sequence or premature shortening of protein
- addition of one base to the DNA sequence which leads to a change in the reading frame of the complementary RNA leading to two of the nucleotides in the protein being changed from what they were supposed to be
Nonsense Mutation
-any genetic mutation that leads to the RNA sequence becoming a stop codon instead
Missense Mutation
- any genetic mutation that changes an amino acid from one to another
- code for a new amino acid after a mutation
Mutations originate at the ___ level but show their effects at the ____ level
- DNA
- Protein
Transcription
- the creation of mRNA from DNA template
- occurs in nucleus of cell with use of DNA dependent RNA polymerase
Template Strand (Anti-sense, Non-Coding Strand)
- one of the single strands of DNA that is read by RNA Polymerase II and acts as the “template”
- the RNA product is complementary to this strand
- these are the 3’ to 5’ strand of DNA
Non-Template Strand (Sense, Coding Strand)
- other single strand of DNA that is not used by RNA Polymerase II
- new RNA transcript from the template strand is nearly identical to this strand except for the presence of ‘U’ instead of ‘T’ on the RNA strand
- these are the 5’ to 3’ strand of DNA
RNA Polymerases
- enzymes that transcribe DNA into RNA
- uses DNA as a template to build a new strand of RNA through base pairing
- reads DNA template strand in 3’ to 5’ direction so that it can synthesize a 5’ to 3’ strand
- doesn’t have the ability to proofread like DNA Polymerase does
- it is DNA dependent
Upstream
nucleotides that come before the initiation site
Downstream
nucleotides that come after the initiation site
List the steps involved in Transcription
- Helicase and Topoisomerase unwind double stranded DNA
- RNA Polymerase II binds to the TATA Box (via assistance from Transcription Factors) within the Promotor region of the gene on the template (anti-sense) strand
- RNA Polymerase moves down the template strand (in a 3’ to 5’ direction) and creates a chain that grows from 5’ to 3’
- RNA Polymerase reaches Stop codon on template strand – form a hairpin after stop codon which causes the RNA Polymerase to fall off
- primary transcript formed from the DNA template is heterogenous nuclear RNA (hnRNA) which later forms mRNA
POST-TRANSCRIPTIONAL PROCESSING - Spliceosome recognizes the 5’ to 3’ splice sites of exons and splices the introns out of the strand then ligates the exons together
- 7-Methylguanylate Triphosphate Cap is added to the 5’ end of hnRNA (pre-mRNA) transcript
- Polyadenosyl (Poly-A) Tail is added to 3’ end of hnRNA (pre-mRNA) transcript
- this processed strand is now mRNA and can exit the nucleus via nuclear pores and undergo translation in the cytoplasm
Alternate Splicing
- process by which more than one mRNA strand can be made from the same gene
- splices out various combinations of introns to create multiple different proteins
- leads to an increase in protein diversity / genetic diversity
RNA Polymerase I
- located in nucleolus
- synthesizes rRNA
RNA Polymerase II
- located in nucleus
- synthesizes hnRNA and some nuclear RNA (snRNA)
RNA Polymerase III
- located in nucleus
- synthesizes tRNA and some rRNA
DNA -> DNA
- Replication
- new DNA synthesized in 5’ to 3’ direction
DNA -> RNA
- Transcription
- new RNA synthesized in 5’ to 3’ direction (template is read 3’ to 5’)
RNA -> Protein
- Translation
- mRNA read in 5’ to 3’ direction
Translation
- conversion of mRNA transcript into function protein
- occurs in ribosomes in the cytoplasm
What is required for translation to occur?
- mRNA
- tRNA
- ribosomes
- amino acids
- GTP
What is the main function of a ribosome during translation?
bring mRNA together with charged aminoacyl-tRNA complex to form a protein
What are the 3 ribosome binding sites?
- A (aminoacyl)
- P (peptidyl)
- E (exit)
- these are found on large protein which contains tRNA
What subunits are Eukaryotic ribosomes composed of?
- 80s ribosome
- composed of an upper 60s subunit and a lower 40s subunit
What subunits are Prokaryotic ribosomes composed of?
- 70s ribosome
- composed of an upper 50s subunit and a lower 30s subunit
What stages of translation require energy (GTP)?
all 3 stages (A, P, and E)
What are the 3 steps that occur during Translation?
- Initiation
- Elongation
- Termination
Initiation
(1) charged initiator tRNA (anti-codon) binds AUG on mRNA via base-pairing within P Site of ribosome – initial amino acid formed is always methionine
(2) GTP hydrolysis and Initiation Factors (not permanently associated with ribosome) then allows large subunit to bind to small subunit
(3) once subunits are bound, tRNA is in P site and have an empty A and E site
Elongation
(1) anti-codon of an incoming aminoacyl-tRNA base-pairs with the complementary mRNA in the A site via aid from hydrolysis of GTP
* Every time tRNA binds, GTP hydrolysis occurs!*
(2) rRNA molecule of large ribosomal subunit catalyzes the formation of a peptide bond between new amino acid in the A site and the carbonyl end of the growing polypeptide in the P site
(3) the polypeptide from the tRNA in the P site is removed and attached to the amino acid on the tRNA in the A site
(4) empty tRNA in the P site is moved to the E site where it is released
(5) the ribosome translocated the tRNA in the A site to the P site
(6) mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site – GTP hydrolysis occurs
- 3 step cycle repeated for each added amino acid
- ribosome moves in 5’ to 3’ direction along mRNA from its amino (N-) to carboxyl (C-) terminus
- Elongation Factors (EFs) locate and recruit aminoacyl-tRNA along with GTP while helping remove GDP
Termination
(1) ribosome reaches a stop codon on mRNA in the A site
(2) termination codon in the A site binds a release factor (a protein shaped like a tRNA)
(3) release factor promotes hydrolysis of the bond between the tRNA in the P site and the last amino acid of the polypeptide, thus freeing the polypeptide from the ribosome P site
(4) the two ribosomal subunits dissociate from each other
What occurs during Post-Translational Processing?
- cleavage of proteins or signal sequences via hydrolysis
- formation of quaternary structure (ex. hemoglobin)
- covalent addition of other biomolecules (ex. carboxylation, phosphorylation, glycosylation, prenylation)
Carboxylation
- addition of carboxylic acid groups
- usually serve as Ca2+ binding sites
Phosphorylation
- addition of PO4 2- by protein kinases to activate or deactivate proteins
- most commonly seen with serine, threonine, or tyrosine
Glycosylation
addition of oligosaccharides as proteins move through ER and golgi to determine cellular destination
Prenylation
addition of lipid groups to certain membrane-bound proteins
Chaperones
specialized class of proteins that assist in protein-folding
Operon Structure
- cluster of genes transcribed as a single mRNA
- either inducible or repressible gene clusters
- offer a simple on/off switch for gene control
Trp Operon
particular cluster in E.Coli
Jacob-Monod Model
- used to describe structure and function of operons
- contain structural genes, operator site, promoter site, and regulator gene
Structural Gene
codes for protein of interest
Operator
- nontranscribable region of DNA that can bind a repressor protein
- like an “on/off” switch
- part of the promoter that is upstream of genes
Promoter Site
- provides place for RNA Polymerase to bind
- within 25 base pairs from transcription start site
What happens when a repressor is tightly bound to operator system?
RNA Polymerase can’t move from promotor to structural gene -> reduction in transcriptional activity
Inducible System
- normally “OFF” but can be made “ON” given a particular signal
- inducer binds to repressor so repressor can’t bind to operator
- increased concentration of inducer leads increased transcription of genes
- Ex. Lac Operon
Lac Operon
- contains gene for lactose
- inducible system
- bonded to repressor under normal conditions but when lactose is present it is turned on by an inducer pulling the repressor from the operator site
- lactose is the inducer because it’s what starts the process of transcribing the genes
Positive Control Mechanisms
- binding of molecule increases transcription of a gene
- inducible systems become this in presence of an inducer
Repressible Systems
- normally “ON” but can be made “OFF” given a particular signal
- allow constant production of a protein product
- repressor made by regulator gene is inactive until it binds to a corepressor which then binds to operator site to prevent transcription
- serves as negative feedback loop: increase in product -> increased in repressor -> increase in complex attachment to operator -> decreased transcription of same gene
- Ex. Trp Operon
Trp Operon
- negative repressible system
- increase in tryptophan acts as a corepressor to bind to operator site and turn off cell synthesis of tryptophan
- Trp is the co-repressor
What are different ways that gene expression can be controlled in Eukaryotes?
- transcription factors
- gene amplification
- regulation of chromatin structure
Transcriptional Factors
- transcription-activating proteins that bind to the TATA Box in the promoter and facilitate RNA Polymerase binding
- have 2 domains: DNA-Binding Domain, Activation Domain
DNA-Binding Domain
- part of transcription-activation proteins
- bind specific nucleotide sequence in promoter region or to response element (DNA sequence that binds to specific transcription factors) to help recruit transcriptional machinery
Activation Domain
- part of transcription-activation proteins
- binds several transcription factors and other regulatory proteins (ex. RNA Polymerase, Histone Acetylases) which function in remodeling of chromatin structure
Gene Amplificiation
- occurs when gene expression must increase in response to hormones, growth factors, or other intracellular conditions
- can occur from enhancers or gene duplication
Enhancers
- grouping of response elements that allow for control of gene expression by multiple signals
- enhancer regions are located >25 base pairs away from transcription start site
How do signal molecules promote gene expression?
- include steroid hormones (cortisol, estrogen) and second messengers (cAMP)
- signal molecules bind to their specific receptors in the nucleus -> these receptors are transcriptional factors that use their DNA-Binding Domain to attach a particular sequence in DNA called a response element -> once bonded to response element the transcription factors promote an increase in gene expression
Histone Acetylation
- leads to an increase in gene expression
- occurs when transcription factors recruit co-activators like histone acetylases which acetylate lysine residues on the amino terminal tail regions of histone proteins whcih decreases the positive charge on lysine residues and weakens interaction of histone with DNA
- leads to opening of chromatin conformation
Histone Deacetylases
remove acetyl groups from histone tails -> leads to closing of chromatin conformation -> decrease in gene expression
DNA Methylase
- enzyme that adds methyl groups to cytosine and adenine nucleotides
- results in silencing of gene expression
What regions of DNA are heavily methylated?
heterochromatin regions
What level is gene expression regulated at?
the level of transcription
In a prokaryotic cell, the special initiator aminoacyl transfer RNA molecule that binds to the start codon transports which amino acid?
Formylmethionine
Sense
-5’ to 3’ strand
Anti-Sense
-3’ to 5’ strand
Degeneracy of Genetic Code says that ___ amino acids are coded for by more than __ codon
- many
- one
AUG codes for ___
methionine
Silent Mutation
- point mutation that results in the same amino acid
- would never know there was a mutation unless looked at genetic code
- mutation usually occurs at wobble position
___ Amino Acids
___ Bases
___ Codons
- 20
- 4
- 64
Upstream is normally ___ numbers
negative
upstream is to the left of whatever is in question
Downstream is normally ____ numbers
positive
downstream is to the right of whatever is in question
What does the 7-Methylguanylate Triphosphate Cap on the 5’ end of hnRNA do?
- helps mRNA move from nucleus to cytoplasm by protecting it from degradation
- consists of a bunch of guanine nucleotides
What does the Polyadenosyl (Poly-A) Tail on the 3’ end of hnRNA do?
-protects mRNA against rapid degradation (composed of 50-250 adenine bases)
Do prokaryotes have transcription factors?
No
TATA Box
- found in promoter of eukaryotes
- about 25 nucleotides upstream of start codon
- where transcription factors bind
What happens during mRNA processing?
splice out introns from pre-mRNA in the nucleus via use of spliceosome
What is a spliceosome made of?
- snRNPS (small nuclear RNA proteins)
- proteins
Splice Donor Site
-located at end of first exon in pre-mRNA
Splice Acceptor Site
-located at start of second exon in pre-mRNA
Are Ribosomes organelles?
NO
What are ribosomes composed of?
- protein
- rRNA
Anti-Codon and Codon are _____
complementary
How do you charge a tRNA? ( steps)
(1) amino acid binds first to enzyme aminoacyl-tRNA synthetase via use of ATP (ATP coupled rxn)
(2) correct tRNA floats over to enzyme and binds
(3) a covalent bond is formed between tRNA and the amino acid which kicks off AMP
(4) aminoacyl tRNA (“activated” amino acid) leaves the enzyme
What enzyme catalyzes tRNA charging?
Aminoacyl-tRNA synthetase
A Site
- “A = Arrival”
- aminocyl-tRNA binding site
P Site
- peptidyl-tRNA binding site
- peptide bond is formed as polypeptide is passed from the tRNA in the P site to the tRNA in the A site which requires enzyme Peptidyl Transferase (found in large subunit), use of GTP during formation of bond
E Site
- “E = Exit”
- this is where we end up having tRNA with no amino acids attached so the tRNA now exits
During translation for every amino acid we add to the peptide, we need ___ ATP/GTP
3
One advantage that prokaryotes have is that they can make ___ at the same time that _____ is occuring.
- mRNA (transcription)
- translation
Do prokaryotes have exons?
No – means also do not have spliceosome
When a protein is in the rough ER, it is usually modified via ____
glycosylation (addition of sugars)
What are operons?
- a cluster of genes that are under the control of the same receptor
- found in prokaryotes only
Coordinated Control
- occurs when multiple genes are under the same promoter
- cellular signals will either turn everything on at the same time or everything off at the same time
In the absence of glucose, ___ levels are high and it will start to bind to CAP
cAMP
CAP= catabolic activity protein
What does the cAMP/CAP complex do?
it binds to the operator and helps RNA Polymerase bind which leads to an increase in the synthesis of the lac operon
What if a mutation in an E.coli cell changes the lac operon so the repressor protein cannot bind?
because the lac operon is always on, there will be synthesis of lac genes without lactose
What if there was a mutation in CAP that increased its affinity for cAMP?
- the synthesis of the lac genes will be increased even in the presence of low cAMP levels
- even if glucose is present the operon may not turn down
What if there was a mutation in CAP that decreased its affinity for cAMP?
there would be a decrease in the synthesis of the lac genes even when there’s no glucose
