Central Dogma Flashcards
Why are new borns given heel prick test
Testing for PKU (Phenyl Alananin Ketonurosa)
PKU
Lack PAH enzyme
PAH (Phenylalanine Hydroxylase) = converts L-Phenylalanine –> L-Tryosine
If lack PAH = can’t convert = Increase in Phenylalanine = causes severe neurological disorders
- Easy to treat – low Phe diet BUT you need to do this before you show symptoms
Central Dogma
DNA –> RNA –> Protein
DNA –> DNA – replication (DNA polymerase)
DNA –> RNA – Transcription (RNA Polymerase)
RNA –> Protein – Translation (Ribosome)
Locations in Central Dogma
Eukryotic DNA = replicated in Nucleus
Transcition = in nucleus
THEN after transvcription = send RNA out of nucleous –> RNA goes to cytosol where the ribosomes are
Tranlastion = In cytosol
Genome vs. Transcriptome vs. proteome
Genome (DNA) – 22,000 genes
Transcriptome – 200,000 transcripts –> All RNAs from the 22,000 genes
- Is transient – DNA is stable BUT RNA is not stable
- Some RNAs are converted to proteins – proteins can be modified after they are made
Proteome – 1 Million proetins –> All proteins in cell
- Some RNA can make 2 different propteins through post transcriptional modifications (includes modified proteins)
Metabolome – Metabolites – entire set of small molecules “metabolies” that are used in the betabolisms
ALL OF THESE = INFLUENCED BY THE ENVIRNMENT
- Interactioned between them = contribute to phenotype
If have the protein –> CAN see what the RNA would look like –> From RNA can see what the DNA would look like
TRP - PHE - GLY - SER
Steps to get DNA:
1. Determine RNA
2. Deduce DNA template
Look at codon table to get RNA
RNA – 5’ UGG-UUU/C-GGN-UCN 3’
-ACU/C
***Can have mutliple choices of codons –> don’t know which it is
THEN can get template DNA
RNA – 5’ UGG-UUU/C-GGN-UCN 3’
Template DNA – 3’ ACC - AAA/G - CCN - AGN 5’
Direction of Ribosome
Reads 5’ - 3’
Example – TRP - PHE - GLY - SER
TRP –> SER
Means – 5’ GGU –> CUU 3’
3’ - 5’ = template strand
5’ - 3’ = coding strand
RNA polymerase = 5’ –> 3’
DNA – 3’ CGT GGT ACC AAA 5’
mRNA 5’ GCA CCA UGG UUU 3’
CODON = GCA – codon on template = produces peptides (LOOK AT IMAGE)
Coding vs. Template strand
dsDNA –> opens to get ssDNA – the ssDNA is read by RNA polymerase to make transcript (RNA)
- RNA = single stranded
Only one DNA strand is transcribed – ONLY one of the two strands is read by RNA polymerase –> Coding strand is NOT read by RNA polymerase
RNA = complementary to template strand (complementary to 3’-5’)
- Template = the strand that is read by RNA polymerase to direct RNA synthesis
RNA = identical to coding strand but has U instead of T (5’ - 3’ strand)
RNA vs. coding strand
mRNA is identical to coding (5’ - 3’ strand) except has U instead of T
Where does RNA polymerase bind
Binds to and transcribes the template strand (3’ - 5’)
mRNA = complementary to template strand
How much of genome is transcribed
Only 5 - 10% of genome is transcribed
- The whole genome us replicated BUT not the whole genome is transcribed
ONLY 2% of the things transcribes have codes that turn into proteins
***Only regions of chromosomes are transcribed to RNA
Types of RNAs
- mRNA – carry protein coding sequnece (2% of RNAs)
- Has information that directs protein synthesis
- RNA in Central Dogma = mRNA
- rRNA – used to build proteins (80% of RNAs)
- Components of ribosomes (Ribozymes involved in protein synthesis)
- tRNA – brings Amino Acids to Ribosomes
- Binds to Amino acids and delivers them to ribosomes to aid in Protein Synthesis
- ncRNAs – Used for gene regulation and/or structure (Direct when transcription and translation occur)
***Most RNAs are not carrying protein information
Transcription
Process of synthesis RNA from DNA template
Stages:
1. Initiation
2. Elongation
3. Termination
What types of RNAs are part of process of making proteins
rRNA + tRNA + mRNA
rRNA + tRNA = part of process of making proteins – involved in converting mRNA into proteins
Transcription initiatian (Prokyrotic)
Sigma factor binds to -10 and -35 elements on the coding strand
When binds it bends dsDNA causing the -10 element to become single stranded
- -10 region is very AT rich = weaker bonds in -10 than -35 = can pop open = open up dsDNA
END: Get ssDNA including transcript start site
**Sigma factor = Subunit of RNA polymerase
**DNA needs to be opened to get single stranded template
How do cells decide where to start transcription
RNA polymerase recognizes promoter sequences in DNA that directs where transcription will begin
Bacteria – have -10 and -35 elements that are positied near +1 site
The sigma domain of RNA polymerase scans the genome looking for promoter sequences –> When at promoter it binds and RNA polymerase is positioned where transcription should start
-10 element (Pro)
10 BP upstream of +1
Concensus sequence = Roch in AT – TATAAT
Promoter Pro
Two regions in core promoter –> -10 and -35 elements
-10 = 10 BP upstream of Start site
-35 = 35 BP upstream of start (TTGACG)
**Promoters have recognition sites for RNA polymerase
**Bacteria core promoter for nearly all genes are similar
***DNA sequences = highly conserved = can build consensus sequences for each –> Can be similar but not identical
Versions of alpha + Sigma factors
Bacterial cells = produce many versions of sigma + Alpha factors that help recognize and bind to specific promoters sequences and UP elements
- Helps cells be selective about which RNAs they will produce at a given time
- Offers one level of gene regulation
Alpha factor
SU in RNA polymerase – has long flexible antena that reach out and bind to up elements
Initiation (pre-class)
RNA polymerase = has many Subunits
Sigma factor Subunit of RNA polymerase
- Sigma = has 2 domains that recognize and bind to -35 and -10 elements
Within sigma factor = have helic-turn-helix sturctures –> Helix-turn-helix domain binds to major groove of DNA near -10 element = causes dsDNA to bend –> bend causes tension in dsDNA –> To relieve the tension dsDNA begins to separate at the AT rich -10 element = allows RNA polymerase to see +1 Start site and initiate transcription
- dsDNA seperates to 2 single stranded regions
ANOTHER SU – Alpha factor = has long flexible antena that reacj out and bind to Up elements
What happens when sigma factor binds
When binds it bends dsDNA causing the -10 element to become single stranded
Causes DNA to bend –> the bend causes dsDNA to open = get ssDNA including transcript start site
Similar to DNA A popping open dsDNA during replication
-10 region nucleotides
-10 region is very AT rich = weaker bonds in -10 than -35 = can pop open = open up dsDNA
Result of Initiation
RNA polymerase opens dsDNA = epsoe coding and template strand –> now have start site in open single stranded bubble
Once RNA polymerase is bound to the promoter and has opened the dsDNA exposing the start site = initiation is complete = move to elongation
+1 site
Where transcription begins (beginning of transcription region) – 1st BP that is read by RNA polymerase
In bacteria – the first nucleotide of RNA transcriot = usually A (Have a T on template 10 BP downstream from -10 element)
Promoter vs. +1 site positions
Promoter sequences occur upstream of transcription start site
Upstream vs. Downstream
Upstream = to the left –> Sequences before the transcription start site
Down stream = to the right –> Sequences after the start site
Example – -35 and -10 are upstream from +1 start site
- Upstream = before + 1
- Downstream = after +1
Example 2 –
-10 = 10 BP upstream of start site
+10 = 10 BP downstream of start site
**Talking about relative position – Relates to the direction that RNA transcription takes place
**Relative position can be provided by +/- #
Set up after intiation
-35 elememnt upstream from +1 (35 BP away from start site – away from +1)
-10 element upstream of +1
+1 – transcription start site
***There is no 0 on the DNA “number line”
First nulcdeotide on RNA transcript (bacteria)
The 1st nucleotode on RNA = A –> Means that it is from T on the DNA strand 10 BP downstream from -10 element
Sigma factor during elongation
RNA polymerase will begin to elongate newly transcribed RNA molecule – as it moves away from promoter = no longer need sigma factor – sigma factor is released where it can go and find a new promoter and RNA polymerase complex
Building of RNA
RNA polymerase builds RNA in 5’ –> 3’ direction building RNA chain in complementary and Antiparallel to DNA template strand
RNA polymerase during elongation
RNA polymerase holds the DNA chain like a fist sliding up a rope
Chanels;
1. dsDNA enters and exits
2. Ribonulceotides enter
3. RNA exit chanel
Center of RNA polymerase has Sununits that act as helicase to seperate the dsDNA
Has Subunit to add nucleotides to growing RNA cgain
Has chaparone to refold DNA into dsDNA
RNA pol + Primer
RNA polymerase does NOT require a primer – can make RNA with now existing chain (can build RNA immediately – just needs template)
- Primase = an RNA polymerase used in DNA replication – primase is the defintion of an RNA polymerase (creates nucleotide chain)
Vs. DNA pol – needs a primer
Answer: D
Just need to look for the most common nucleotide in each position
Concenus sequence
Most common nucleotide at each position
- Concencus sequence is a representation of aligned sequences where each nucleotide (of amino acid) represents the most common one at that position
- The average nucleotide sequences when comparing a number of similar sequences
Don’t need them to all have the same just needs to be the most common
***-10 and -35 = concenus sequnces –> promoter might not be identical
Example concenusus
Genes in E.coli – have -10 and -35 region –> they are similar but not identical
6 out of 7 = have T in teh forst two positions of -35 region = Consenus is TT
3rd position – 4 have G, 2 have T, 1 has a C – Concenus is most frequently observed = G
Overall: means that promoters do NOT need to be identical to concensus sequence – -10 and -35 elements are numbers by the avergage position of middle element
- Actual position of the -35 for a gene might not be exactly 35 BP downstream fo start site – might be centered at -34 or -30 BUT by convention we still refer to as -35
Core promoter
-10 and -35
UP elements
Need gene regulation in transcription –> can have region of genome upstream of -10 and -35 that can interact with RNA polymerase for regulation
Can have additional regulatory sequences that are upstream of core promoter
Can be located anywhere 40-250 BP upstream of +1
***Do not exist for every gene AND when they do exist they are very varaible (no obvious concensus sequence_
NOT essential (Not like core promoter that is needed) – if it is mutated transcription still occur BUT at a much lower level
Elongation (Prokaryots)
RNA polymerase wraps around the DNA
There is a hole in Polymerase for incoming DNA + a hole for outgoing DNA (has a channel to pump out DNA) – refolds DNA to leave + has chanel for nucleotides otp come in + Has a chanel for RNA to leave
Polymerase ALSO acts like a Helicase
RNA polymerase structure
Overall: large Enzyme with many Subunits that helps with activities
2 entry chanels – DNA and rNTP chanels
2 exit chanels – DNA and RNA exit chanels
RNA polymerase acts as a helicase unzipping the dsDNA chain AND it acts as a chaperone to help zip the dsDNA back together after it has been transcribed
ANSWER: FALSE – gioes until there is a signal to stop RNA
What strand does transcription occur on
Can occur from either strand of DNA – can go on both strands
- Typically look at sequences where the top strand is 5’ - 3’ – IF we see the promoter the RNA is syntehsized in the fowards directing using that strand as a template BUT the promoter on can be on opposite strand = read in opposite direction (STILL 5’ - 3’) but have -10 and -35 on that 5’ - 3’ direction = transcription occurs in reverse direction
- Genomes are big – genes occur on both the top and bottom strands of DNA
-35 and -10 are upstream of +1
Template = 3’-5’
Coding = 5’-3’
THE CODING = also can have -35 and -10 – can just flip around and use that strand
- Can go on the coding side (from the bottom
IMAGE – shows places where transcription is occur
RNA polymerase as a helicase
RNA polymerase acts as a helicase THEN refolds dsDNA and sends out
Why not stop transcription at stop codon
Wouldn’t stop transcription at stop codon because there are other types of RNAs – the process needs work for all RNAs
Termination Transcription Process (Bacteria)
Occurs when palindromic termination sequence in DNA is transcribed into ssRNA –> ssRNA does not want to stay single stranded – wants to form douvle stranded = palindrome will snap together and form dsRNA = creates stem loop structure
The stem loop in RNA is bulky – now you have a bulky thing behind RNA polymerase = RNA polymerase can’t hold onto template anymore = RNA polymerase falls off DNA
Answer: B – won’t know where to stop
Termination in Transcription - Rho Dependent Vs. Rho Independent
BOTH = involve a stem loop structure
Rho Independent:
Just requires Termination sequence
- Have stem loop and RNA polymerase falls off
Rho Dependent:
The palindrome can form unstabel stem loop (BUT THIS STEM LOOP IS MORE UNSTABLE – might be shorter or more AT rich) – the stem loop is not enough to interfere with RNA polymerase = Polymerase can get through the stem loop (because unstable) = have a rut sequence uptream from termination sequence –> When Rut sequnece is transcribed RNA is recognized by Rho factor – Rho binds to RNA and goes in the same direction as RNA polymerase and Rho is faster than RNA polymerase –> Rho can catch up to polymerase
- The palindrome goes out of the exit of polymerase and Rho comes to stabilize the stem loop = have a stable stem = RNA polymerase falls off
Answer: C – RNA polymerase can keep going
Transcription can be…
Continuous – as Polymerase moves off of promoter a new RNA polymerase can come in and start even before the 1st polymerase is done the 2nd can come in
- Once RNA polymerase has moved off the promoter a new Polymerase can bind and initiate a 2nd transcript
IMAGE – see DNA in the middle and the strands coming off are RNA being made – as go down DNA strand RNA is longer
- Shows RNA transcripts emerge from transcribed regions of DNA
- Center = dsDNA
- Things coming off = RNA
- Can deduce position of promoter from image – RNA strands that are closer to promoter will be shorter than those farther from the promoter = shorter = begining
ANSWER: B – The start has shorter RNA; Farther = get longer
Synthesis of all RNAs
All RNAs are synthesized in similar ways – BUT not all RNAs carry protein coding information
Sites in Ribosomes
Ribosome has 3 sites for tRNA binding:
5’ E P A 3’
E – Exit Site
P – Pepityl Site – Amino acid chain on tRNA in the P site gets transfered to Amino acid on tRNA in A site
A – Aminoacyl site – tRNA is initially brought to A site
- Where new tRNA comes in
tRNAs
Short RNAs (80 Nucleotides) that brings Amino Acids to ribosome
- Always the same shape
Shape – 3 stem loops
- AA is on the acceptor end
- Part that recognizes the codon = stem loop on the bottom using Anticodon
General Structire of mRNA after transcription
Have:
1. 3’ UTR region – region upstream of coding sequence – NOT translated
- 5’ UTR – NOT translated –> Has termination sequence from transcription
- Ribosome binding site – upstream of coding sequence – recognized and bind to ribosome
- Open reading frame = Coding sequnce – THIS IS THE PART THAT IS TRANSLATED
- Starts with AUG
- Ends at Stop codon
- Organized into groups of 3 nucleotides (Codons)
Translation Inititaion (Prokaryotes)
Goal: Build the ribosome onto the mRNA
16S rRNA (on small SU) binds to ribosome binding site – 1st codon AUG is just downstream of this
- Annals to the complimentary sequence in the ribosome domain – BINDS TO SHINE DELGARNO SEQUENCE
rRNA comes and binds to ribosome binding site –> THEN another protein called initiator factor comes in –> When comes in now the small SU is assembled
THEN bring in 1st Amino Acids (F-Met) –> binds with initiator THEN 2nd initiator binds (second initiator binding uses GTP – requires Energy)
***Uses GTP – requires energy to build
END = 30S SU (Initiation factors + Small SU + GTP + F-Met
THEN Large Subunit comes on (Once already have F-met) – Puts F-met in the P Site
- IF 1, 2,3 are released and the large SU binds to produce the 70S initiation complex
At the end – the ribosome is assembled on the mRNA and the first tRNA is attached to the initiation codon in the P site of the ribsome
End of translation initiation (Prokaryotes)
At the end – the ribosome is assembled on the mRNA and the first tRNA is attached to the initiation codon in the P site of the ribosome
ALL TOGETHER = 70S
Translation Elongation (Pro)
A chargered tRNA is deleivered to the ribsome by an elongation factor + engery (charged tRNA enters A site)
EF-Tu + GDP is then release (can be reused)
THEN the amino acid in the P-site tRNA is attached to the amino acid on the A site tRNA – creates peptide bind
THEN the ribosome moves downstream to shift the unvcharged P site tRNA to the E site (where it is released) AND the tRNA that was in A is now in P site with the Peptide chain
THEN a new charged tRNA is recruited to the A site – process repeats until reach stop codon
ANSWER: FALSE
ONLY translates after ribosome binding site + until stop codon
Translation Termination
A release factor is recruited to the stop codon –> causes the ribosome to disassemble and fall off the mRNA and the protein chain to be released
- At the stop codon = bring release factor = block –> ribosome stops and falls off
Operons
A cluster of Open reading frames (genes) that are transcribed together on one mRNA (one promoter + one terminator)
- Bacteria = can have mRNA with multiple reading frames (cluster of genes on one mRNA = operon)
- Each coding sequence = has its own ribosome binding site -> ribosome finds site = attaches and makes proteins
- One template
***Has multiple ribosome binding sites
Prokaryotic transcription + translation
Can occur simultaneously – as soon as ribosome binding site is transcribed = ribosome can come in and begin translation before transcription ends
- Ribosomes will be moving towards the RNA polymerase
- Transcription + translation go at the same rate
Where does splicing occur
Splicing of introns occurs in the nucleus
Alternate splicing
Can make multiple proteins from the same gene
- Common in Eukaryotes
Example – Allows 20,000 genes in humans to create 400,000 proteins
Structure of Eukaryotic mRNAs
End – End with Poly A tail
- Poly A tail = Polyadenylation sequence
- At 3’ end
- Have 100-200 A nucleotides at 3’ end of Eukaryotic mRNAs
Beginning - have 5’ Cap
Eukaryotic Translation - Initiation
The ribosome is recruited to Euk
mRNAs with the help of initiation factors called CAP binding proteins that bind to 5’ and 3’ poly A tails of mRNA
***Initiation tRNA carries Met NOT f-Met
