Transcription and Translation (Part 2)

in eukaryotes, where does transcription happen, and what needs to happen before translation

• transcription happens in nucleus, and transcript must be processed before it can exit nuclear pore and go into cytoplasm with ribosomes where translation happens

how is the path of transcript different in prokaryotes

• in bacteria, mRNA doesn’t need processing, ribosomes can actually even begin translation while transcription is still taking place, and no nucleus

what is the first procesing step in euk transcript as transcription is taking place actually

• addition of 5’cap

what is the 5’ cap

GTP or guanosine nucleotide

in what orientation is the 5’ cap added

• 5’ - 5’ orientation, so like added to 5’ end, but it’s end is also 5’

when is the 5’ cap added

• during transcription

what adds the 5’ cap

• capping enzyme attached to RNA pol II

what process happens to cap and by who

• cap is methylated by

• methyl transferase

why is 5’ cap needed

• efficient translation and stability

• so protecting mRNA from degredation

• and recriting translation machinery

what is needed to be done to transcript after 5’ cap is added, and what is the process called

• 3’ poly tail is added

• called polyadenylation

what exactly is added where in polyadenylation

• 50-250 adenines, As, are added to 3’ end of RNA

what are the 3 purposes of the 3’ poly a tail

• stabilize mRNA from exonucleases that break down RNA

• interact with 5’ cap to position ribosome

• signal for export of mRMA from nucleus

what is the 1st step in the 2 step process of polyadenylation

• makes 3’ end by cleavage of mRNA at ~11-30 bases downstream of a consensus sequence in mRNA

what is the consensus sequence for step 1 in polyadenylation

• AAUAAA

what is the final and second step in polyadenylation

• adenines added by poly A polymerase

what causese transcription termination in euks

• polyadenylation

for termination, the RNA transcript needs to be broken for the 3’ poly tail to be added, where is it broken

cleavage site

what binds and cuts transcript at cleavage site

• a complex protein machinery

what happens to the 5’ end that is left after the cleavage site is broken

it binds to Rat1

what is Rat1

• a 5’ to 3’ exouclease

what does Rat1 do once it binds to the 5’ end of the RNA transcript part that’s still being made but no longer needed

• degrades the tail until it reached RNA polymerase II to stop transcription

ok, so now pre-mRNA is terminated and has 5’ and 3’ cap/tails, what is the step that needs to take place before translation

• mRNA splicing

what are exons

• sequences found both in the genomic DNA and mature RNA (so expressed regions)

• protein coding but also some untranslated regions

what are introns

• sequences found in the genomic DNA but not in the mature mRNA

• they interupt the coding regions and are not protein coding themselves

• most eukaryotes have them

what are the 5’ and 3’ UTRs

• they are the untranslated regions at the very ends of the transcript, but they actually remain in the mature mRNA and have important regulatory roles and help with mRNA stability and translation

what is there at the beginning of an intron

• 5’ consensus sequence which will be at the 5’ splice site of that intron

what is there at the end of an intron

• 3’ consensus sequence which is at the 3’ splice site,

what are 5’ and 3’ consensus sequences usually like

• they ususally follow some recognizable pattern

what is there in the middle of the intron

• a branch point

• which is an adenine residue that helps remove the intron

what actually cuts out the introns and how

• spliceosome cones and cuts out introns by recognizing splice sites

so ultimately, what happens to introns and what to exons/coding regions

• Introns are taken out of mRNA

• Exons/coding regions are translated

what combine to form the spliceosome

• snRNPs combine with proteins

what are snRNPs

• small nuclear ribonucleoproteins

• made up of RNA and protein

where does the spliceosome bind

• at the consensus site

what does spliceosome result in

• spliced mRNA

• and a lariat formation in intron fragment

what is lariat

• loop in intron basically

ok just listing how spliceosome works

• snRNPs help recognize consensus sequence and branch point and allow spliceosome to assemble on mRNA

• spliceosome first leads to a lariat in the intron, which is able to form due to the A in the branch point

• then spliceosome joins exons

• lariat will be broken down and recycles on its own

more introns is associated with what

greater complexity of organism

what does it mean that greater complex is associated with more introns

• means they are not useless, they probably have regulatory roles, so like more introns - more potential regulaion and more opportunities for alternative splicing, which leads to different transcripts and different proteins sometimes

intron size varies a lot too, with more complex animals generally having

• longer introns so probably more regulatory area

what is an extreme splicing example

• human dystrophin gene underlying duchenne muscular dystrophy

• 
  

what was the length of the primary transcript versus final mRNA in the human dystrophin gene

• primary transcript was ~ 2,500,000 nucleotides, which is largest known human gene

• mRNA was ~14,000 so lost about 99% after splicing

what is alternative splicing and what does it lead to

• different mRNAs can be spliced different way for the same transcript or pre-mRNA

• basically results in variations in mRNA, which leads to variations, deletion, additions, etc proteins

alternate splicing gives rise to alternate proteins called what?

isoforms

what is trans-splicing, and is it common in humans

• uncommon in humans

• combines exons from different RNAs

what are the 5 types of splicing we looked at

• consitutive splicing

• exon skipping

• mutually exclusive exons

• alternative 5’ donor site or splice site

• alternative 3’ acceptor site or splice site

what is constitutive splicing

• normal splicing as you would expect

what is exon skipping

• legit just skipping one exon when splicing

what are mututally exclusive exons

• when only one of the two exons can be included in the mature mRNA

what are alternative 5’ donor or splice cites and alternative 3’ acceptor or splice sites

• bascially introns have different splice sites at either the 5’ or 3’ ends, which results in changes in boundary of exon, so added or removed nucleotides

what is an example of tissue-specific isoforms, and what are tissue specific isoforms

• tissue specific isoforms are like when same gene leads to different experession but based on tissue or cell type

• gene is the alpha tropomyosin gene

what can mutations in introns affect

• intron splicing and RNA splicing in general

what is an example of an intron mutation leading to issues

• hemophilia B mutation in european royals

• mutation in the factor IX gene is in the intron, makign a new 3’ splice site which leads to change in mRNA and thus nonfunctional clotting factor

is there any correlation between number of genes, so genome size and complexity of an organism

NOOO

is there any correlation betwen complexitiy of organism and amount of alternative splicing

YESS positive relationship

what percentage of genes in humans show alternative splicing

• 95%

translation is moving from the language of what to the language of what?

from language of nucleic acids to the language of amino acids

how many potential nucleotide bases are there versus amino acids

• 4 potential nucleotide bases

• but 20 possible amino acids

if there was just one base translating to amino acids, how many amino acids would we be able to translate

4, not enough

what if there were 2 bases translating to each amino acid, how many amino acids would we be able to translate

• 4 possibilities in first base, 4 in second base, so 4 × 4 = 16

• still not enough

what if there were 3 bases translating to each amino acid, how many amino acids would we be able to translate

• 4 possible bases for 1st base, 4 for second, and 4 for third, so 4×4×4 = 64

• enough!!

what is the genetic code that translates nucleotides to amino acids called and why

• called degenerate code

• because have more codons (or base base base combos) than 20 amino acids

• so 64 codons are coding 20 amino acids

nucleotides are read in codons which are

• 3 base non-overlapping units

how does translation show the importance of getting the template strand right

• because getting template strand wrong leads to 2 different amino acids, even if you like flip it, still doesn’t match

so in what direction are codons read

• 5’ to 3’

what is the impact of code degeneracy

• multiple DNA sequences can encode the same protein

• for example 1152 different DNA sequence combinations can code the same 7 amino acid sequence

what is the reading phase

• like the way you read codons, which will be different based on where you start

shifting starting point of reading codons by a single nucleotide does what

• shifts how all codons are read, which can make a HUGE difference

• called a phase shift

how to decide which reading frame is correct

• always need to start with AUG codon, (Met) and always end in stop codon

likely proteins will have now many amino acids between AUG and stop

~ 50, if it doesn’t fit these requirements, some shift likely took place

what are 3 exception to the genetic code being fully universal, making it only nearly universal genetic code

• often stops change and code for amino acids

• a lot of changes, like stops coding for amino acids in mitochondria

• other changes for use of modified amino acids

what is the mechanism of translation

• polypeptide synthesis

what are the needed things for polypeptide synthesis in translation

• mRNA template

• transfer RNAs (tRNAs) and amino acids

• Ribosomes (ribosomal RNA and proteins)

  • enzymes that catalyze reaction

what are transfer RNAs or tRNAs and what do they do

• adaptors that connect mRNAs to correct amino acids

• they are short single stranded RNA

tRNAs are able to have correct shape and do correct job because they

• have many modified bases

which part of tRNA pairs with correct codons

• anticodon pairs with complementary nRNA codons

amino acids are attached to which end of tRNA

• 3’ end

what is the amino acid attachment site on the 3’ end always made of

CCA

if the amino acid attachment site is fixed, what allows corect amino acid attatchment

• the enzyme aminoacyl-tRNA synthetase

how does aminoacyl-tRNA synthetase work

• loads amino acid onto correct tRNA recognized by sites on all arms of tRNA

there is a different what for each amino acid

• different aminoacyl-tRNA synthetase

what is it called when tRNA is attached to amino acid, and what is abbreviation

• charged tRNA

• abbreviation tRNA^XYZ

tRNAs being attached with correct amino acid is one of most important steps why?

because if synthetase enzyme doesn’t do this step correctly, ribosomes don’t check that tRNAs have correct amino acid attached, so wrong amino acid will just go right into translation

codon itselt if in what direction, and what direction is anticodon in

• codon is read in 5’ to 3’, and thus anticodon will be 3’ to 5’

what is wobble position

• third pair of codon pairs less strictly with 1st base of tRNA anticodon so single tRNA can recognize multiple mRNA codons

what does wobble position explain

• that there are actually less than 61 tRNAs in most animals because 61 would be necessary (no need for stop codons) but instead have less because of wobble position

how many tRNAs in humans and how many in E.coli

• 50 tRNAs in humans

• 47 tRNAs in E.coli

what are the ribosomes the site for

• protein synthesis

what are the two types of ribosomes location-wise, both make proteins, just proteins will end up in different places

• free in cytoplasm

• attached to endoplasmic reticulum

what do ribosomes from cytoplasm do

make proteins for cytoplasm

what do ribosomes on ER do

• make proteins to be secreted and placed in membranse, and shipped to different organelles

what type of structures are ribosomes, and what are they made of

• complex structures

• made of rRNA and protein

what are two subunits or prokaryote ribosomes

• 50S subunit (prokaryote specific name) - large subunit

• 30S subunit (prokaryote specific name) - small subunit

what does the 50S subunit do?

• responsible for catalyzing reactions that make peptide bonds between amino acids

what does the 30S subunit do

• reads mRNA and finds right position to start

what are the stages of bacterial translation

• initiation

• elongation

• termination

what does initiation in bacterial cells require?

• mRNA and two subunits of ribosome

what is the first step in bacterial initiation of translation

• small subunit binds mRNA at the Shine-Dalgarno sequence, especially in bacterial cells it’s this sequence specifically

so what does small subunit binding to Shine-Dalgarno help do

• helps position ribosome close to start codon, which is very important for having right reading phase

better match between mRNA and ribosome in the shine-dalgarno sequence means what

higher translation