Genetics Exam 3

coding RNA

mRNA only

non-coding RNA

tRNA, rRNA, snRNA, siRNA, miRNA

mRNA

directly encodes for proteins, carriers genetic information from DNA to the ribosome for protein translation

tRNA

brings amino acids to the ribosome during translation and matches them to the mRNA codon via its anticodon

rRNA

structural and functional component of ribosome; catalyzes protein synthesis

snRNA

involved in splicing within the spliceosome; helps process pre-mRNA into mature mRNA

siRNA

involved in RNA interference leading to mRNA degradation and gene silencing

miRNA

regulates genes expression by binding to mRNA and inhibiting translation or promoting degradation

Structure of RNA

single stranded for flexibility and folding

have an -OH group on the 3' carbon which makes it more unstable because the groups ability to break phosphodiester bonds

contains uracil which bonds with adenine and also guanine

sense strand

coding strand/non-template strand

gene sequence is the same as mRNA sequence

runs in the 5' to 3'

antisense strand

non-coding strand/template strand

RNA polymerase reads this strand to create an mRNA transcript that is complementary

runs in the 3' to 5'

-35 region

consensus sequence TTGACAT that is recognized by RNA polymerase

-10 region

consensus sequence TATAAT that helps RNA polymerase know when to start unwinding DNA

5'-UTR

untranslated region between promoter and coding region

prokaryotic initiation

1. RNA polymerase binds to promoter with help of sigma factor

2. DNA unwinds to form transcriptional bubble

3. RNA polymerase starts synthesizing at the +1 site

4. sigma factor is released once transcription starts

prokaryotic elongation

1. RNA polymerase moves along the DNA template from 3' to 5'

2. Synthesizes mRNA in the 5' to 3' direction (NTPs added to 3' OH)

3. Adds ribonucleotides to growing strand

4. DNA rewinds behind the RNA complex as it moves forward

5. continues until termination sequence is identified

where does energy for transcription come from?

hydrolysis of NTPs and the breakage of phosphodiester bonds

prokaryotic termination

RNA polymerase continues after the coding region and forms 3' UTR, transcription ends when RNA polymerase encounters a signal

factor-independent termination

occurs after a GC rich sequence followed by a A rich sequence

1. GC rich region forms a stem loop structure in RNA which prevents backtracking by RNA polymerase

2. RNA polymerase pauses after transcribing U-rich sequence

rho-dependent termination

1. RNA has a RUT upstream of a polymerase pausing site

2. Rho binds to the RUT site and moves along the RNA toward the 3' end

3. When Rho reaches paused RNA polymerase, it unwinds the RNA-DNA hybrid causing RNA to be released

Rho

a helices that unwinds nucleic acids

nuclease

any enzyme that degrades nucleic acids

exonuclease activity

degrading from the end of the nucleic acid, digested RNA from the 3' end

endonuclease activity

cuts within a nucleic acid, cuts mRNA into peices

mRNA decay importance

allows bacteria to quickly alter gene expression in response to changing nutritional and environmental conditions

prokaryotic transcription

-polycistronic

-no post-transcriptional processing

-no nucleus

-transcription and translation occur simultaneously

eukaryotic transcription

-monocistronic

-post-transcriptional G-cap and poly-A tail

-DNA is packaged into chromatin and wrapped around histones

-splicing occurs to remove introns and connect exons

-genes are spaced farther apart

exons

coding regions

introns

intervening non-coding regions

pulse-chase experiment

An experiment in which cells are grown in radioactive medium for a brief period (the pulse) and then transferred to nonradioactive medium for a longer period (the chase).

RNA pol I

transcribes rRNA genes (except 5S) that form the structure of ribosomes and are essential for protein synthesis

RNA pol II

transcribes all protein coding genes (mRNA); important for producing template for protein synthesis

RNA pol III

transcribes other small RNA genes (tRNA and 5S rRNA); produces essential RNA for translation

RNA CTD

-carboxyl terminal domain

-unique tail-like region on the large subunit of RNA polymerase II

-plays critical role in coordinating RNA processing during transcription

CTD function

-regulates initiation by recruiting general transcription factors and the mediator complex

-needed for capping, splicing, and polyadenylation

phosphorylation

promotes elongation

dephosphorylation

signals termination and RNA release

eukaryotic initiation

1. TFIID (TBP) binds TATA box

2. Other TFs + RNA Pol II form the Pre-Initiation Complex (PIC)

3. TFIIH unwinds DNA & phosphorylates RNA Pol II

4. RNA Pol II starts transcription & releases most TFs

general transcription factors

recruit RNA pol II to promoter

TFIID

first to bind to promoter, contains TBP

TBP

binds to TATA box, recruits pre-initiation complex

TATA box

-30 bp, binding site for TBP

DPE

downstream promoter sequence, initiates transcription

TFIIH

phosphorylates CTD, binds to promoter sequence to initiate transcription

gene specific transcription factors

regulate RNA pol II activity by turning on or off specific genes in response to cell signals

eukaryotic termination

RNA pol II relies on a Poly U stretch which destabilizes the RNA DNA hybrid and leads to dissociation

5' G Capping

-occurs immediately after transcription initiation when RNA is ~25bp long

2. CTD of RNA Pol II recruits enzymes responsible for capping

3. GTP is added to the 3' OH end, then a methyl group is added to form 7-methylguanosisne (m7G)

4. necessary for protecting RNA from degradation, splicing, and aiding in translation initiation

3' Poly-A tail

-added immediately after the cleavage of the pre-mRNA near the end of transcription

- PAP adds 5-250 A bps to the 3' OH end

- protects from degradation, aids in transcriptional termination and release of RNA pol II, promotes translation

splicing

-spliceosome removes introns from the primary transcript

-U1 binds the 5' splice site (GU), U2 binds to the branch point

-U4, 5 and 6 join and form complex

-U1 and 4 are released from spliceosome then the first splice is created at GU

-second splice at AG and then exons are lineked

-occurs co-transcriptionally

alternative splicing

helps to produce multiple protein variants from one mRNA, can allow for greater protein diversity without increasing the number of genes

TREX export

binds to mRNAs 5' cap with help of CAP-binding complex during transcription and directs it to the nuclear pore to go to the cytoplasm

PHAX export

-snRNAs are transcribed and capped at the 5' end

-CBC binds the 5' cap and recruits PHAX

-PHAX escorts the snRNA out of the nucleus through a nuclear pore

-snRNAs associate with proteins to form snRNPs

-mature snRNPs are then imported back into the nucleus for spliceosome assembly

mRNA half life

from 20-30 minutes to 20-24 hours after the removal of the polyA tail, decay occurs in both directions

mutations

common change from adenosine to inosine that can pair with A, U or C; allows multiple proteins to be translated from the same gene

holozyme

-core enzyme and its required cofactors or subunits

-RNA polymerase holozyme has a core RNA polymerase and the sigma factor

sigma factor

protein subunit that associated with RNA polymerase in prokaryotes to enable specific promoter recognition and transcription initiation; different factors recognize different promoter sequences

consensus sequence

a sequence of DNA or RNA that represents the most common nucleotides found at a particular position, often in promoters

(Ex: -35 and -10)

snRNPs

small nuclear ribonucleoproteins; key in RNA splicing by recognizing splice sites in pre-mRNA and are essential components of the spliceosome

spliceosome

a large protein-RNA complex responsible for removing introns from pre-mRNA in eukaryotic cells and also ligating exons together

RNAi

-mechanism for gene silencing by promoting RNA decay

-gene specific

-involves miRNA and siRNA

RNAi mechanism

-detects bad signal and uses a helper protein, Dicer, to chop up bad regions into tiny pieces called siRNA

-another protein, argonaute, grabs a peice of the siRNA and looks for matching pieces in the cell to cut up

-miRNA regulates the good mRNA, assists argonauts with searching

-RISC is powered by argonaute and uses siRNA and miRNA as a template to find matches to load up

-overall helps to stop harmful proteins from being made

primary structure

chain of amino acids linked by peptide bonds

secondary structure

hydrogen bonds between amino acid backbones causing the chain to folding into two common shapes- alpha helices, and beta pleated sheets

tertiary structure

protein folds into a complex 3D shape by stabilizing side chains

quaternary structure

protein complexes made up of multiple polypeptides (ex: hemoglobin)

hydrophobic side chain

avoid water and stay inside

hydrophilic side chain

attract water and stay on the outside

charged side chains

form ionic bonds and stabilize

cysteine

forms disulfide bridges and stabilizes

prokaryotic ribosome

50S + 30S = 70S

eukaryotic ribosome

60S + 40S = 80S

rRNA role in ribosome

-most abundant RNA

-some have structural roles others enzymatic

-make up over 50% of ribosome

-transcribed and assembled into the ribosome in the nucleolus

tRNA structure

-anticodon loop with three nucleotides that are complementary to a codon on mRNA

-amino acid holder on 3' end (5'-CCA-3') where aminoacyl-tRNA synthetase attaches the correct amino acid

-anticodons are written 3' to 5' and codons are written 5' to 3'

tRNA charging

-aminoacyl-tRNA synthetase uses two proofreading steps to minimize error when adding the correct amino acid

-binds amino acid and ATP molecule, ATP is hydrolyzed and releases PPi

genetic code

-mRNA is read from 5' to 3' in triplet code

-continuous and non-overlapping

-same for almost all living organisms

-multiple codons can code for the same amino acid (degeneracy)

-start: AUG

-stop: UAA, UGA, UAG

-64 total codons

G pairs with

C or U

U pairs with

A or G

inosine (I) pairs with

A, C or U

tRNA wobble

third base on codon and first base on anticodon is more flexible and allows tRNA to recognize multiple codons

Prokaryotic translation initiation

1. IF3 binds to the small subunit to prevent large subunit from joining early

2. mRNA binds and aligns with Shine-Dalgarno with AUG in the P site

3. IF1 binds to the A site, blocking early tRNA entry

4. !F2 bound to GTP recruits Fmet-tRNA to P site

5. GTP hydrolysis triggers release of IFs

6. Large subunit joins and translation begins

** happens at the same time as transcription

prokaryotic translation elongation

1. a new tRNA enters the A site, matching its anticodon with the mRNA codon

2. Ribozymes form a peptide bond between the amino acids in the P and A sites

3. Growing polypeptide transfers to the tRNA in the A site

4. ribosome moves one codon forward in the 5' to 3' direction

5. the tRNA in the P site moves to the E site and leaves

6. tRNA in the A site moves to the P site and it all repeats

prokaryotic translation termination

Ribosome reaches a stop codon and no tRNA binds, release factors bind instead and trigger cleaving of the polypeptide and disassembly of ribosome

eukaryotic translation initiation

1. eIF1, eIF1A, and eIF3 bind to the small subunit to prevent early binding of large subunit

2. eIF2 bound to GFP binds to met-tRNA, forming the ternary complex

3. small subunit and ternary complex combine to form pre-initiation complex

4. eIF4F complex recognizes the 5' cap and PABP binds to the 3' Poly A tail to circularize the mRNA

5. Pre-initiation complex scans along (5' to 3') the mRNA for the start codon

6. eIF5 stimulates GTP hydrolysis causing the release of eIFs and the recruitment of the 60S large subunit

** occurs only after mRNA processing and transport to cytoplasm

eukaryotic translation elongation

1. EF-Tu brings in charged amino acids

2. Peptide is added to the amino acid in the A site

3. EF-G pushes the ribosome along the mRNA one codon at a time

4. Energy for both peptide bond formation and ribosome movement come from GTP hydrolysis

eukaryotic translation termination

1. no tRNA bind to stop codons

2. 3 release factors bind to stop codons

3. hydrolysis results in the removal of the polypeptide chain

Prokaryotic translation

-30S small subunit binds Shine-Dalgarno, positioning AUG in the P site, IFs bring first tRNA and then large subunit joins

-uses Formylmethionine

-polycistronic

-does not use poly a tail

eukaryotic translation

-pre-initiation complex assembled with met-tRNA (non-formulated) recognizes 5' cap and brings in the small subunit, slides down, then large subunit joins

-monocistronic

-uses a poly a tail to aid in initiation

nonsense mutation

when a stop codon appears too early in an mRNA sequence stopping protein synthesis before the full protein is made, results in a short and nonfunctional protein

suppressor mutation

surpasses the effect of an earlier nonsense mutation by either:

1. a mutant tRNA recognizes the premature stop codon and inserts an amino acid instead of stopping translation

2. A mutation in another gene can help compensate for the loss of the original protein

co-translational sorting

-ensures proteins destined for secretion, membranes, or organelles enter the correct cellular compartments during translation

-Signal sequence on protein directs the ribosome to the ER

-Signal recognition particle binds to the signal sequence and pauses translation

-SRP then binds to the SRP receptor on the ER membrane and translation continues

Signal transduction

-allows cells to respond to external signals such as hormones, by transmitting messages inside the cell

-a signaling molecule binds to a receptor on the cell surface, receptor changes shape or gets phosphorylated

-activated receptor activates signaling proteins inside the cell that amplify the signal

Ubiquitination

-marks proteins for degradation or alters their function to regulate cell processes

-protein is tagged with ubiquitin, a small protein that marks it for degradation using E1, 2, and 3 enzymes

-proteasome recognizes ubiquitinated proteins and unfolds/degrades them into small peptides

-small peptides are broken down into amino acids for reuse

post-translational sorting

-synthesized in the cytoplasm and sorted to mitochondria, nucleus, peroxisome, and chloroplast (in plants)

-targeting sequences within the protein direct it to the appropriate organelle and chaperone proteins help unfold and transport the protein

-imported into the organelles through complexes and then refolds and functions

RISC

RNA-induced silencing complex

-multiprotein complex that mediates gene silencing by using siRNA or miRNA to bind to complementary mRNA sequences, leading to mRNA degradation

Dicer

an enzyme that cleaves double-stranded RNA or pre-miRNA into siRNAs or miRNAs, which re then loaded onto RISC for gene silencing

Argonaute

a key protein in the RISC complex that binds to siRNA or miRNA and facilitates mRNA cleavage or translational repression

reverse genetics

-a method used to study gene function by starting with a known gene sequence and then disrupting or modifying it to observe the resulting phenotype

-seeks to find what phenotypes arise as a result of particular genetic sequences

-ex: RNAi, transgenic mice, clone and alter

N terminus

NH2; start of a protein

C terminus

COOH; end of a protein where new amino acids are added

condensation reaction

carboxyl end of one amino acid reacts with the amino group of another amino acid and a peptide bond is formed with water released as a byproduct