bio 305 exam 4

pre-discovery of mRNA

• pulse-chase method was used to track newly synthesized RNA, using radioactive nucleotides

• Volkin and Astrachan studied transcription in bacteria after infection by a bacteriophage. . .they saw that radioactivity from uracil broke down quickly, so translation involves a type of RNA with short lifespan

• subsequent experiments also showed that radioactivity was concentrated in the nucleus and lingered for a short time in the cytoplasm, indicating that the RNA was likely an intermediary

discovery of mRNA

• Brenner, Francois, Jacob, and Meselson did an experiment to determine whether new phage protein synthesis in bacteria needed newly constructed ribosomes or existing bacterial ribosomes

• experiment found that existing ribosomes are used to produce phage proteins, and RNA that directed this protein synthesis formed and degraded quickly

• this allowed them to conclude that phage “messenger” RNA with a short half-life is responsible for protein synthesis during infection

snRNA

small nuclear RNA, found in nucleus of eukaryotic cells, participate in mRNA processing and intron removal

miRNA and siRNA

recently recognized types of regulatory RNA that are active in animal and plant cells, important for controlling stability or translatability of certain mRNAs. . .implicated in gene regulation

4 stages of transcription

1. promoter recognition and identification

2. initiation of transcript synthesis

3. transcript elongation

4. transcript termination

upstream

near 5’ start of transcript

downstream

near 3’ end of transcript

promoter

nucleotide sequence that’s not transcribed, a transcription-regulating DNA sequence that controls access of RNA polymerase to the gene

bacterial RNA polymerase

• only one bacterial RNA polymerase (E. coli), came from experiment with antibiotic rifampicin

• five-polypeptide RNA polymerase core that binds to 6th polypeptide (sigma subunit) that has a conformational change in core enzyme into active form (holoenzyme)

• sigma subunit allows core enzyme to bind specifically to promoter sequences

sigma factor

RNA pol subunit in prokaryotes that is only involved in initiation

alternative sigma subunits

alter specificity of holoenzymes for promoter regions by imparting distinct conformational changes to the core

consensus sequences

short regions of DNA sequences that are highly similar, though not necessarily identical

structure of prokaryotic protein coding genes and mRNAs

DNA

—(-35)———(-10)—(+1, transcription start)———————————————(terminator sequence)

mRNA

(5’ UTR)[start codon]———coding sequence———[stop codon](3’ UTR)

pribnow box sequence

-10 consensus sequence consisting of 6 bp 5’ TATAAT 3’ and separated by 25 bp from another 6 bp region (-35) 5’ TTGACA 3’

UTR

untranslated mRNA

how does RNA polymerase holoenzyme initiate transcription? (prokaryotes)

1. initial loose attachment to double-stranded promoter sequence and then binds tightly to it to form closed promoter complex

2. bound holoenzyme unwinds about 18 bp of DNA around -10 consensus sequence to form open promoter complex

transcription elongation

• when the holoenzyme reaches 1+ nucleotide, it beings RNA synthesis using the template strand

• holoenzyme remains intact until first 8-10 nucleotides have joined, then sigma subunit dissociates from core enzyme, which keeps on going. . . sigma subunit can go to another core enzyme to transcribe another gene

• DNA is unwound and then rewound after the enzyme passes

end product of transcription

ssRNA that’s complementary and antiparallel to template DNA strand

transcription termination mechanisms

• usually signaled by DNA termination sequence containing repeating sequence producing distinctive 3’ RNA sequences

• intrinsic and rho-dependent termination (less frequent)

intrinsic termination

• two features: inverted repeat and string of adenines in template DNA beginning at 5’ end of inverted repeat 2 region (3’ end of mRNA)

• transcription of inverted repeats makes mRNA with complementary segments that fold into short ds stem ending (hairpin)

• string of uracils complementary to adenines follow haripin structure on 3’ end of mRNA

• those things cause RNA polymerase to backtrack to hairloop and destabilize, until it falls off DNA and transcript is released

rho-dependent termination

• less common, requires activation of rho protein to bind to new mRNA and catalyze separation of mRNA from RNA polymerase to terminate transcription

• rho utilization site transcription produces rut site on mRNA, where rho protein attaches and moves towards RNA polymerase

• when RNA polymerase reaches terminator sequence hairpin, rho protein can catch up and catalyze release

order of information flow, aka the “basic assumption”

DNA ↔ RNA —> protein

protein is last stage of flow, can’t go back from that

DNA can recreate itself (replication) and so can RNA, protein can recreate itself in some cases (like prions)

dogma

a principle or set of principles laid down by an authority as incontrovertibly true. . .may not always be the case, and could be detrimental in a science. . . Dr. Wierzbicki calls them the enemy of knowledge

unique quality of RNA

its function and structure is in between DNA and proteins. . .

• it can carry genetic info in nucleotide sequence like DNA

• can form complex structures and carry out biochemical functions, like proteins (can act as enzymes)

compare and contrast prokaryote and eukaryote transcription/translation

prokaryotes:

• circular genome

• DNA transcribed to mRNA and mRNA translated to proteins at same time

eukaryotes:

• linear genome

• DNA transcribed into mRNA in nucleus

• mRNA processed and transported

• mature mRNA translated to proteins in cytoplasm

three RNA polymerases in eukaryotes

RNA pol I, RNA pol II, RNA pol III

RNA polymerase I

transcribes several ribosomal RNA genes

RNA polymerase II

transcribes mRNA that encodes polypeptides, also snRNA

RNA polymerase III

transcribes all tRNA genes and one snRNA and rRNA gene

memorization hack for eukaryotic RNA polymerases: RooMaTe

RooMaTe

R is first in mnemonic, RNA polymerase I is for rRNA

M is second in mnemonic, RNA polymerase II is for mRNA

T is third in mnemonic, RNA polymerase III is for tRNA

some exceptions. . . RNA pol II and III both make snRNA also, and RNA pol III also makes one rRNA gene

structure of eukaryotic protein coding genes and mRNAs

DNA

—(TATA-box)—(+1, transcription start)————————————————(polyA signal)

mRNA

cap(5’ UTR)[start codon]———coding region———[stop codon](3’ UTR)AAAAA

TATA box, aka goldberg-hogness box

• most common eukaryotic promoter consensus sequence

• approx position -25 to +1 start fo transcription

other consensus eukaryotic promoters

CAAT box, GC-rich box, OCT box, etc.

eukaryotic RNA polymerase II holoenzyme

14 subunits, two largest subunits form catalytic site

preinitiation complex for transcription

composed of RNA pol II, TFIID, general transcription factors (GFTs), and template DNA

general transcription factors (GFTs)

TFIIA, TFIIB, F, E, H, J

very important in telling RNA polymerase where to start transcribing

transcription factors

proteins that bind to promoter regulatory sequences and influence transcription initiation by interacting (directly or indirectly) with RNA polymerase II

TFIID

multisubunit protein containing TATA-binding protein (TBP) and subunits of TBP-associated factor (TAF) that binds to TATA box, separates strands of DNA to start transcription

fate of preinitation complex after initiation

most factors unbind, TFIID remains at promoter to help another RNA pol II molecule, while RNA pol II continues after initiation

CTD

• C-terminal domain

• not structured domain

• can be phosphorylated, there is a distinct pattern of phosphorylation for each stage of polymerase

transcription termination in eukaryotes

• once RNA pol II reaches polyadenylation signal, a ribonuclease cleaves RNA and poly-A polymerase makes a polyA tail to keep mRNA stable

• polyA is template-independent, the polyA tail is not coded for by DNA

RNA processing in eukaryotes

5’ capping, splicing, and poly-adenylation

what coordinates RNA processing?

• CTD, through its phosphorylation pattern

• cotranscriptional process, no unprocessed RNA floating around

5’ cap

7-methylguanosine, backwards GTP in 5’ to 5’ connection

hnRNA

heterogenous nuclear RNA, immature and unprocessed RNA with exons and introns

what is RNA splicing?

removal of introns

what is responsible for splicing?

• spliceosome, a ribonucleoprotein complex with snRNPs (snRNA and protein) and additional proteins

• it can determine splice donor and acceptor sites, we can’t really do that yet

splice donor site

dinucleotide GU, 5’ splice site

slice acceptor site

dinucleotide AG, 3’ splice site

describe the splicing process

1. spliceosome is assembled

2. first splicing reaction: cleaving at splice donor site, spliceosome attaches to branch site

3. second splicing reaction: cleaving at splice acceptor site, exons join and intron removed as lariat, which will eventually degrade

what is the most practical way to find intron location?

isolate cDNA and do sanger sequencing

alternative splicing

• one gene can give rise to more than one polypeptide

• in human genome, about 25000 genes give rise to about 100000 proteins

process of mitochondrial transcription

long transcripts that are almost whole genome —> cleavage into individual tRNA, rRNA, and mRNA —> regulation of RNA stability occurs for each fragment individually

what is used for mitochondrial transcription?

phage-type single subunit polymerase (unusual)

3 transcripts produced from mtDNA

H-strand (heavy) and 2 L-strands (light)

snoRNA

small nucleolar RNA, does rRNA processing

what is the function of transcription initiation?

defining the 5’ end of mRNA

enhancer sequences

DNA regulatory sequences that increase level of transcription of specific genes. . .usually located upstream of genes they regulate but can be downstream as well

silencer sequences

DNA sequences/elements that repress transcription of genes, binding proteins that bend DNA so that genes are hidden from transcription activation by RNA pol II

heterochromatin

densely packed chromosomes

euchromatin

loosely packed chromosomes. . . “u” for uncoiled, or “e” for expressed, meaning transcribable!

features of promoters recognized by RNA pol I

• core element, -45 to +20

• upstream control element, -100 to -150

• both are rich in G and C

function of 5’ cap

1. protecting mRNA from rapid degradation

2. facilitating mRNA transport across nuclear membrane

3. facilitating subsequent intron splicing

4. enhancing translation efficiency by orientating ribosome on mRNA

polyadenylation signal sequence

AAUAAA

torpedo model of transcription termination

• specialized RNase acts as a torpedo aimed at residual mRNA attached to RNA pol II, because it has no cap protecting the 5’ end

• it quickly destroys the residual mRNA left after cleaving the mature mRNA from RNA pol II and catches up to RNA pol II, causing it to dissociate from the template DNA and end transcription

branch point

20-40 nucleotides upstream of 3’ splice site, containing a branch point adenine. . .critical for accurate intron removal

self-splicing introns

introns that can self-catalyze their own removal, ribozymes that are classified into group I or group II

rRNA processing

rRNAs are transcribed as large precursor molecules that are cleaved into smaller RNA molecules, denoted in S units (svedberg)

tRNA processing

• some produced simultaneously with rRNAs, others transcribed as part of large pre-tRNA transcript that’s cleaved to make multiple tRNAs

• nucleotides are trimmed off at 5’ and 3’ ends to prepare

• tRNAs fold into 3D structure looking like a clover, with three hairpins and one stem

• AA binding site is added to 3’ end (CCA terminus)

what is the direction of translation?

5’ to 3’

corresponding mRNA and protein structural landmarks

N-terminal corresponds to 5’ end, C-terminal corresponds to 3’ end

ribosome tRNA sites

A - accepts new AAs

P - protein polymerizes

E - polypeptide exits ribosome

3 essential tasks of ribosomes

1. bind mRNA and identify start codon where translation begins

2. facilitate the complementary base pairing of mRNA codons and tRNA anticodons that determines amino acid order in the polypeptide

3. catalyze peptide bond formation between AAs during polypeptide formation

ribosome structure

• consists of 2 main subunits, large and small, measured in svedberg units (S)

• large subunit has enzymatic activity (ribozyme) and has 3 catalytic sites. . . E, P, and A

E. coli ribosome

small subunit is 30S, large subunit is 50S, fully assembled is 70S

mammalian eukaryotic ribosome

small subunit is 40S, large subunit is 60S, fully assembled is 80S

cryo-EM

• cryo-electron microscopy, pioneered by robert glaeser in 70s and perfected jaques dubochet in 80s

• uses liquid nitrogen or ethane to instantaneously freeze macromolecules and preserve them in their native state

where is the anticodon on tRNA, location where codons bind to?

‘clover’ end directly opposite 3’ end with AA attachment site

aminoacyl-tRNA synthetase

• enzyme specific to each AA (20 total) that recognizes both AA and tRNA

• matches correct AA to mRNA codon

translation initiation in prokaryotes

• the small subunit, together with initiation factors (IFs) bind the Shine-Dalgarno sequence, near AUG start codon

• a charged (AA-bound) tRNA binds

• large ribosomal subunit binds

• IFs release

what is the initiator tRNA charged with?

fMet for prokaryotes, Met for eukaryotes

Shine-Dalgarno actual sequence

typically AGGAGGU

translation initiation in eukaryotes

• small subunit with eukaryotic initiation factors (eIFs) binds 5’ cap, scans mRNA for AUG start codon

• charged initiator tRNA binds

• large ribosomal subunit binds

• eIFs released

kozak sequence

consensus translation initiation sequence in eukaryotes

5’ ACCAUGG 3’

what provides energy for assembly of ribosome and translation machinery, as well as steps of elongation?

GTP

polycistronic

• mRNA in prokaryotes

• multiple polypeptides produced from a single transcript due to multiple Shine-Dalgarno sequences and multiple translation initiation sites

monocistronic

• mRNA in eukaryotes

• a single polypeptide produced from a single transcript, because the 5’ cap is the only ribosome entry site and there is only one translation initiation site

translation elongation steps

similar in prokaryotes and eukaryotes, but eukaryotes have more complex and numerous protein factors

1. entry of tRNA with bound AA into A site, aided by elongation factor (EF)

2. ribosome catalyzes peptide bond formation, with newly formed dipeptide in A site

3. ribosome movement along mRNA causes dipeptide to move to P site and uncharged tRNA from P site moves to E site and is released

4. repeat!

translation termination steps

1. release factor (RF) binds stop codon

2. catalytic activity of RF makes polypeptide dissociate from tRNA

3. tRNA and mRNA separate from ribosome

4. ribosome dissociates into large and small subunits

features of genetic code, as demonstrated by experiments

• number of codons per AA is variable

• three stop codons

• universal

• no overlaps or gaps between codons

• degenerate code, more than one code per amino acid, so the third nucleotide isn’t as important

what can the different genetic codes be understood as?

same information in different languages

evidence for triplet nature of genetic code - crick and brenner 1955

• used proflavin, a molecule that distorts DNA’s double helix and can cause insertions and deletions

• created mutations in rII gene

• one or two insertions or deletions caused frameshift mutations that change output, but three insertions or 3 deletions (or same number of both deletions and insertions) cause no change

evidence that codons are nonoverlapping - Heinz Fraenkel-Conrat 1960

• if overlapping - one alteration can affect 3 consecutive codons. . . if nonoverlapping - one alternation affects one codon

• used nitrous oxide, which caused a mutation of a single nucleotide

• found that only one codon/amino acid was altered, so codons are nonoverlapping!

how were codons assigned to amino acids? nirenberg, matthaei, khorana

• nirenberg and matthaei created synthetic mRNAs with specific sequences (like polyU or polyA) and did in-vitro translation

• khorana developed synthetic mRNAs with repeating di-, tri-, and tetranucleotide patterns, which revealed even more

• last piece as nirenberg’s experiment with mini mRNAs (one codon) and radioactively labeling one of 20 AAs, and they filtered the mixture to only capture mRNA/tRNA/ribosome complex, and were able to match codon to AA for all combos

codon bias

• frequency of synonymous codons differs in various species

• different codons for the same AA have different expression levels across species

what do you need to do if you want to express a protein from one species in another?

optimize the genetic code to get the highest level of translation! choose synonymous codon with the highest expression level

wobble position

bases don’t line up precisely in the 3rd position. . .one nucleotide can base pair with more than one other one

by convention, which strand in dsDNA is coding and which is template?

top —> coding, sense

bottom —> template, anti-sense

polyribosomes

busy translational complexes containing multiple ribosomes that are each actively translating the same mRNA

synonymous codons

codons that specify the same amino acid