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mRNA is derived from

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much larger hnRNA (preRNA)

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introns

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first identified '77 when split genes were discovered

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3' UT contains what additions?

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sequences that control mRNA stability, translation via small regulatory RNA (miRNA) binding

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functions of 5' cap and 3' polyA tails (4)

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resistance to degradation
transport out of nucleus
translation initiation
mRNA stability and turnover

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polyA also used for

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pulling out mRNAs, and cloning mRNA (cDNA cloning)

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splicing sites

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AG GU(A/G)AGU.....A.....[py tract]AG G...

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precision of mRNA splicing is directed by

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small nuclear RNAs that bind highly conserved sequences in introns at exon/intron boundaries

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first transesterification reaction

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1. cleavage at 5' exon/intron junction
2. lariat formation

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second transesterification reaction

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1. cleavage at 3' exon/intron junction
2. 5' exon --> 3' exon splicing

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composition of spliceosome

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5 snRNPs (small nuclear ribonucleoprotein particles (snRNPs, called “SNURPs”)
uridine-rich small nuclear RNAs; U1, U2, U4, U5, U6 snRNAs
~150 proteins

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roles of snRNAS in splicing

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U1 binds 5’ splice site, U2 binds intron branch site A
U4-U5-U6 complex brings 5’ site & branch site A together
U6 catalyzes 5’ splice site cleavage & lariat formation
U5 mediates 3’ splice site cleavage & 5’ exon joining to 3’ exon
snRNP functions carried out by spliceosome uridine-rich snRNAs, rather than proteins

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one gene--> multiple mRNAS

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alternative splicing: Alternative splicing generates different mRNAs from the same pre-mRNA,
This produces different proteins from a single eukaryotic gene

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where was alternative splicing first discovered?

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antibody heavy chains, adenovirus

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2 types of alternative splicing and explain:

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constitutive: 2 or more splice variant mRNAs always made
regulated: splice variant mRNAs are made in only certain cell types or at certain times of development, different tissue-specific patterns of splice variant mRNAs often occur in different species

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what % of human genes are alternatively spliced?

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70

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list stop codons:

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UAA, UGA, UAG

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3 rules that govern the genetic code

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Codons in mRNA are read 5’ --> 3’
Codons are non-overlapping and there are no gaps between codons in mRNA
mRNA is translated in a fixed reading frame set by the initiation or start codon (e.g., AUG)

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All tRNAs have nearly identical 3-dimensional structures that differ
significantly in only two places

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-the anticodon loop
-the 3’ acceptor stem (including
discriminator base)
Recognition of these two regions by specific aminoacyl tRNA synthetases -> charging tRNA with
correct amino acid

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2 step reaction requiring ATPs in charging of tRNAs

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1. ATP adenylylation -> activation of amino acid
2. transfer of amino acid to CCA-0H of tRNA

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tRNA synthetases

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One tRNA synthetase for each amino acid (20 synthetases)
Same synthetase charges all tRNAs that carry the same amino acid (called isoaccepting tRNAs)

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accuracy of protein synthesis is dependent upon...

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tRNA synthetase recognition and charging of correct tRNA

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tRNA structures recognized by synthetase:

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acceptor stem (major determinant) with discriminator base (4th base from 3’ end of CCA in acceptor stem) & anticodon loop

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G on tRNA

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C or U

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C

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G

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A

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U

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U

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A or G

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I

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U, A, C

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Proteins are Synthesized From the N-terminus to the C-terminus

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The amino terminus of the amino acid to be added attacks the carboxyl C of the growing polypeptide chain, so that the polypeptide is now covalently attached to the new tRNA

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what do you need for protein synthesis??

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ribosomes
protein translation factors: like IF, EF, RFs
Euk: more factors, same functions
GTP hydrolysis: conformational change
ATP: peptide bond formation (from tRNA charging)
rRNAs

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A-site

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amino-acyl tRNA will enter at this site

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P site

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new peptide bonds are formed at this site with amino-acyl tRNAs

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E site

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transiently occupied by uncharged tRNA that is leaving ribosome

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translation initiation: prok vs euk

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prok: RBS that bind to 16s rRNA, initiation at nearby AUG, used for initiation at internal mRNA sites. (polycistronic)
euk: initiation at AUG after 5' cap, on mRNA, no internal initiation (monocistronic)

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Two types of tRNAMet present in bacterial cells recognize AUG codons

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tRNAMet used to incorporate methionine within the growing protein chain
tRNAiMet used to initiate protein synthesis
In bacterial cells, MET- tRNAiMet is further modified by the addition of a
formyl group
After protein synthesis the formyl group is frequently removed,
often the Met is removed as well

N-formylmethionyl-tRNAiMet
Resembles peptide bond -
Presence of formyl group
allows binding directly to P site

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stop codon is recognized

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on A site by protein termination factors

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RF trigger

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hydrolysis of polypeptide from tRNA in P site, completed protein released from ribosome

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RF 1

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recognizes UAG and UAA

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RF2

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recognizes UGA and UAA

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RF3

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stimulates release of RF1 and RF2

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stop codons recognized by...

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Stop codons recognized by 3 aa “anticodon” peptide
sequences in RFs (via RF protein/mRNA interaction)

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how are eukaryotic mRNAs held in circles?

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proteins that are held at 5' cap and 3' poly A interact, efficient formation of pre-initiation complex with 40s subunit

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3 steps of viruses

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attachment to host cell, penetration, and release of viral genes, or injection of viral genes
viral replication
release of progeny cell virus from infected cell

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plaques

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Each plaque originates from a single cell infected by one virus
Plaques develop from the release of virus & infection of surrounding cells repeated for many cycles
All viruses in a plaque are identical to the parental virus and constitute a clone.

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lysogeny

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infection
lambda repressor allow the viral genes to not replicate self yet... genome inserted into host cell genome. then lysogenic growth

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4 ways viruses work and examples...

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transform normal cells into tumor cells (retroviruses, HPV)
lytic infection (adenovirus, flu, polio)
persistent infection (hepatitus)
latent infection (herpes, HIV)

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how viruses replicate own genes

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(ss or ds DNA or RNA)
dsDNA use host enzymes for replication
RNA must encode their own enzymes. (some carry in virion)
reverse transcriptase (RNA --> DNA)
RNA directed RNA polymerase

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reverse transcriptase route

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reverse transcriptase: RNA--> cDNA (AZT can block this)
DNA replication (ddi can block this)
integration into cell DNA
T-cell activation, provirus transcription
viral RNA
Either viral RNA packaged into virions or translated into HIV polyprotein,
then proteases produce HIV viral proteins (prohibited by protease inhibitor)

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influenza virus

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Eight different single-stranded RNA molecules (segmented RNA genome) in helical nucleocapsid
Nucleocapsid enveloped in lipid bilayer (cell membrane) with viral & cell glycoproteins on surface
Non-uniform virion shape due to lipid membrane envelope derived from infected cells
Viral protein spikes protrude from envelope. Two surface virion proteins, hemagglutinin (H) & neuraminidase (N) define flu strains (e.g., H5N1, H1N1, etc) & are critical for virus infectivity

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new flu strains rise via two mechanisms

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Mutations acquired during genomic viral RNA segment
replication (Recall that RNA polymerases do not have proof-reading functions). Mutations generally account for the minimally-altered flu virus strains that occur seasonally. However, mutations resulting in only 1-2 amino acid changes
in avain H1 changed the 1918 avian flu virus into the pandemic strain able to efficiently-infect human cells.

2. Reassortment of genomic viral RNA segments - occurs
when two different flu viruses infect & replicate in the
same host cell (e.g., co-infection of avian & human flu
viruses) -> swapping of RNA genome segments during virion assembly - can produce new virulent flu strain with ability to efficiently-infect human cells. Pandemics are often due to such reassortments which produce major shifts in flu virus virulence and/or host specificity

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pandemics vs epidemics

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Mutations acquired during genomic viral RNA segment
replication (Recall that RNA polymerases do not have proof-reading functions). Mutations generally account for the minimally-altered flu virus strains that occur seasonally. However, mutations resulting in only 1-2 amino acid changes
in avain H1 changed the 1918 avian flu virus into the pandemic strain able to efficiently-infect human cells.

2. Reassortment of genomic viral RNA segments - occurs
when two different flu viruses infect & replicate in the
same host cell (e.g., co-infection of avian & human flu
viruses) -> swapping of RNA genome segments during virion assembly - can produce new virulent flu strain with ability to efficiently-infect human cells. Pandemics are often due to such reassortments which produce major shifts in flu virus virulence and/or host specificity

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flu pandemics when 3 things occur by mutation or reassortment

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1. New flu virus strains emerge from animal hosts by acquiring the
capability to infect humans
2. New flu virus is exceptionally virulent for humans (most are not)
3. New flu virus is able spread efficiently among humans (human- to-human transmission) (most do not)

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potential next flu pandemic strain

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H5N1
1. H5N1 first appeared in chickens in 1997 in Asia, now seasonally infects &
kills poultry, wildfowl & some mammals worldwide
2. First human fatality in 2003 - thro 2008 has caused ~230 confirmed
human deaths worldwide (has exceptionally high mortality rate, ~60%)
3. To date, H5N1 strain is ineffective in human-to-human transmission
(likely because it infects deep in lungs & thus it is not spread by
coughing/sneezing (usual means of human transmission)

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Avian H5N1 has many lethal features of the 1918 pandemic H1N1 Flu virus

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1. Infections occur deep in lungs & other tissues
2. High lethality due to aberrant immune responses (cytokine storms)
3. Young adults most susceptible (median age of fatal cases = 20 years)
4. H5N1 still exhibits inefficient human-to-human transmission but could
easily acquire this lethal capability

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the 1918 flu caused prologned severe inflammations (cytokine storms) that produced its unusual mortality

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Survey Analyzing Expression of 40 Cytokine & Pro-inflamatory Genes
Microarray data showing that both the reconstructed 1918 pandemic flu strain & a non-pandemic current flu strain (K173) increased expression of pro-inflamatory immune response genes (as shown by red bars) after 3 days infection. Most of these genes returned to normal or lower levels
(as shown by black & green bars, respectively) after 8 days infection by the non-pandemic K173 flu strain, but majority of these pro-inflamatory & immune response genes remain dangerously active (red) with the pandemic 1918 flu virus

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RNA genomes and mutations

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RNA genomes are more prone to accumulate mutations because reverse transcriptases & RNA replicases do not proofread as they synthesize DNA --> important consequences for resistance to anti-viral therapies, origins of mutant viruses & diseases

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modification enzyme

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methylase: The modification enzyme (methylase) adds methyl groups to nucleotides in this specific sequence on both DNA strands - such methylated DNA is resistent to cutting by the restriction enzyme (a.k.a. restriction endonuclease)

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if not methylated.. what happens?

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restriction enzyme cleavage

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most restriction sites

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have short inverted repeat sequences (palindromes)

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what are plasmids

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Circular, double-stranded DNA (sizes 1 - 200 kb)
Occur naturally in bacteria, yeast, higher eukaryotes
Exist in a parasitic or symbiotic relationship within their host cells
Replicate separately from host cell’s chromosomal DNA due to presence of plasmid DNA replication origin (ori)
Contain ancillary genes useful to plasmid host cells
(e.g., genes encoding toxins, antibiotic resistence, heavy metal resistence, restriction/modification enzymes)

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plasmids must have these 3 parts

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restriction sites (unique cloning sites), selectable marker, and origin of replication

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Introduction of recombinant DNA-vectors into host cells is called

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transformation

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versatile plasmid cloning vectors contain

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polyliker w/ multiple unique restriction sites for inserting DNA fragments

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cDNA

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complementary DNA from mRNA
cDNA library can contain cloned DNA copies of all mRNAs expressed in a cell
only exons are represented in DNA clones: open reading frames from mRNA
Since only ~2% of genome is expressed in mRNA, cDNA libraries eliminate ~98% of genomic DNA sequences - cDNA libraries have greatly reduced complexity compared to genomic DNA libraries (this feature reduces number of clones in screening)

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cDNA libraries made from different cell types

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cDNA libraries made from different cell types contain clones of tissue-specific mRNAs that carry out the specialized functions in such cells

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oligo-dTs?

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they're reverse transcriptase that makes cDNA from mRNA. they're hybridized to polyA as primer for cDNA synthesis. first strand synthesis

then initial product is DNA mRNA duplex, then mRNA is degraded so you have single strand cDNA

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second DNA strand synthesized

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using random short oligonucleotide primers, DNA polymerase, and dNTPs

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what happens to blunt ended cDNA?

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attach restriction site linkers to it, digest linkers and ligate sticky ends into plasmid vector, transform E. coli

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ligation of restriction fragments. how does this happen?

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Ligation of Restriction Fragments with Complementary Sticky Ends via T4 Ligase - used for inserting DNA into a vector to make recombinant DNA

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what do you do when you got your recombinant plasmid?

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mi e. coli with plasmids in presence of CaCl2, heat pulse. culture on nutrient agar plates containing ampicillin.
then cell multiplication
then colony of cells, each containing copies of the same recombinant plasmid.

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assembling a genomic DNA library in phage lambda 5 steps

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1. Cleave genomic DNA to ~25kb fragments by partial digestion with Sau3A1 (vGATC)
2. Remove replaceable central region of λ phage genome by BamHI cleavage (GvGATCC)
3. Ligate λ arms to insert genomic DNA (i.e., ~25kb insert DNA fragments isolated from partial Sau3A1 cleavage)
4. Package recombinant λ DNA into phage heads in vitro
5. Infect E. coli -> screen plaques

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what you need for PCR reactions

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primers: short synthetic oligonucleotides complementary to known DNA sequences flanking specific region to be amplified.
polymerase: taq DNA polymerase, isolated from thermophilic bacteria. or Pfu DNA polymerase.

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PCR cycle

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DNA denaturation at 95 degrees, primer annealing at 50 degrees
primer elongation at 72 degrees

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The Most Difficult Part of Recombinant DNA Cloning is Identifying Clones Containing the Gene of interest
3 diff ways to identify and isolate clones in DNA libraries of phage plaques or bacterial colonies

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1. Select for expression of cloned gene in a mutant cell
background (most commonly used in bacteria or yeast), called
Complementation (functional replacement of mutant gene)
2. Purify the protein & use it to identify the gene (a.k.a. reverse genetics) - two general methods:
-specific antibody probe used to screen for desired protein in recombinant DNA expression library
-synthetic DNA oligonucleotide probe set designed from protein coding sequence (based on codon degeneracy) used to screen for desired clone in DNA library by hybridization
3. Screening of recombinant DNA phage or plasmid library using either cloned gene or cDNA, enriched mRNA, or PCR product as radioactive labeled probe in hybridization

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reverse genetics approach 1: antibody screens, require expression of insert DNA

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Use recombinant expression vectors that contain a promoter & linked bacterial protein-coding gene (e.g.,
bacteriophage vectors are useful because expressed
recombinant fusion protein is released in plaques
Cloned DNA inserts, usually cDNA (without introns) or prokaryotic genomic DNA, are inserted in-frame into the coding sequence of the bacterial protein-coding gene present in the expression vector
DNA insert is transcribed and translated in cells to produce “chimeric” fusion proteins present in the recombinant phage plaques (or bacterial colonies)
Plaques (or colonies) are screened with specific antibody or other molecules known to bind to the

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antibody screens

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bacterial lawn, overlay nitrocellulose filter, remove filter, proteins bind to nitrocellulose
incubate w/ filter w/ primary antibody. wash filter.
incubate filter w/ radiolabeled secondary antibody
antibody identifies specific plaques. autoradiography and xray film

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reverse genetics approach II:

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synthesize DNA probes based on amino acid sequences of isolated peptides from purified protein.
using genetic code to synthesize DNA probe set containing all possible coding sequences for a peptide

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fully degenerate probe

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DNA probe set containing all possible coding sequences derived from genetic code is most reliable choice for screening

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screening libraries using labeled DNA or RNA probes in hybridization. examples of hybridization probes

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enriched mRNA (often used in early work before cloned DNA probes became available)
cloned cDNA or gene (in early studies, cDNA clones often isolated first & then used for genomic gene isolation & characterization)
because of high sequence conservation of genes in evolution, cloned probes from different species can be used in screening to isolate a homologous gene or cDNA (e.g., use of mouse globin cDNA for isolating human globin cDNA or gene)
PCR product (now preferred method) due to ready

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microarrays

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measure genome-wide changes in transcription in diff cells or tissues:
developed to quantitate and compare mRNA levels by hybridization to DNAs displayed in high density patterns suitable for microsensor scanning.
use fluorescent-labeled RNA or DNA probes.
rapid and reproducible large scale surveys comparing expression patterns of known genes.
now expanded to analyze all types of RNA transcripts expressed from entire genome (tiling arrays)

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sanger or dideoxy sequencing

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chain termination in presence of dideoxynucleotides. it blocks addition of incoming nucleotides
four template-primer extension reactions carried out w/ substrates for DNA synthesis. You need dNTPs, DNA polymerase, and one ddNTP per reaction. samples denatured and separated on polyacrylamide gel. bands visualized by autoradiography.

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shotgun (random cloning) strategy

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7x sequence coverage conducted to insure all sequences are captured and correctly assigned in final assembly. 3 types of libraries made from isolated human chromosome. small to big plasmid libraries, small ones produce 6 fold genome coverage.
then you assemble sequence into chromosome strings

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bioinformatics

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new science to analyze and compare genomic sequences

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annotation

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identification of all coding, non-coding and regulatory sequences in a genome

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gene finder

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programs detect protein-coding gene sequences: look for ORF, exons/introns, splice sites, polyA

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BLAST

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comparisons of protein sequences predict relatedness and function

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ENCODE

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(Encyclopedia of DNA Elements) next “big biology” project: initiated after human genome finished in 2003, combines computational & experimental strategies to annotate (i.e., define the functions) all expressed sequences in the human genome! ENCODE Analyses of 1% of human genome completed mid-2007.

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comparisons of genomes with biological complexity

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genome sizes, but not gene numbers increase in relation to biological complexity in multi-cellular eukaryotes.

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what are only class of genes notably expanded in mammals?

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defense and immunity

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when genome size increases...

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introns and intergenic sequence increases. biological complexity. repeated sequences (like transposons too)
alternative RNA splicing

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most sig diff b/w chimps and humans result form

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gene duplication/loss

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sites involved with development only in humans

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MYH16: mutant myosin gene found only in humans (limits jaw development, larger brain size)
HAR2: gene regulatory site controlling wrist/thumb fetal development, allowed for increased dexterity for tool making and use

ASPM, MCPH1, CENPJ: four protein coding genes known to control brian size b/c genetic mutations cause microcephaly in humans
HAR1: short non-coding RNA that differs in 18118 bases in humans vs chimps. known to function in human fetal neuron formation during development of cerebral cortex at 2-5 months in human embryos.
AMY1: enhanced starch digestion, allowed diet of higher energy foods

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RNA interference/RNA silencing

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non-coding RNAs play a major role in controlling gene expression and genome stability

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repeated sequences

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different families of repeated sequences have similar but not identical DNA sequences.

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transposons

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make up 10% of human genome. remnants from past infections by DNA viruses or retroviruses. usually stably-maintained in genome, express non-coding RNA. movement of transposons can disrupt genes. genetic disorders.

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pseudogenes

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highly-mutated, non-functional relics of genes

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RNAi again

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cell defense against RNA virus infections. anti-viral cytokine. some viruses make RNAi to combat anti-viral responses of infected cells.
apaptosis, development, preventing movement of transposons, silencing, inhibition of translation, mRNA degradation)

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drosha

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makes 2 cuts to release 65070 nt pre-miRNA containing 21-23 bp base-paired upper stem, with some mismatched bases and single stranded terminal loop from nuclear pri-miRNA.
drosha-cleaved pre-miRNA then exported to cytoplasm and undergoes dicer cleavage.

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RISC

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RNA induced silencing complex formation

dicer cleaved double stranded miRNA or siRNA incorporated into a cytoplasmic RISC protein complex, one strand is removed or degraded, leaving single-stranded "guide" RNA that base pairs to target mRNAs.

Guide siRNA or miRNA base-pairing specifies
RISC recognition of target mRNA - RISC
proteins from Argonaute Family either cleave
target mRNA (argonautes with slicer activity)
or repress translation (those without slicer
activity)

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3 main functions of RNAi

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1. endonucleolytic mRNA cleavage
2. translation inhibition
3. chromatin remodeling -> inhibition of transcription in nucleus.

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single stranded "guide" miRNA or siRNA in mature RISC base-pairs to target mRNA. two outcomes

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mRNA cleavage, carried out by argonaute protein w slicer endonuclease activity in RISC
inhibition of protein synthesis, RISC association w cytoplasmic P bodies (RNA processing sites for mRNA turnover, contain enzymes for de-capping and de-adenylation of exonucleases) -> degradation of mRNA.