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177 Cards in this Set

  • Front
  • Back
Building block of Nucleic acids
nucleotides
2 types of nucleic acids
DNA and RNA
5 main nucleotides used in nucleic acids
Adenine and Guanine which are purines

Cytosine, Thymidine (DNA), and Uracil (RNA) are pyrimidines
3 parts of nucleotide
pentose sugar

phosphate group

on one prime carbon of pentose sugar there is a purine or pyrimidine base
2 major differences between DNA and RNA
1. DNA is DEOXY, it has a H on the 2' Carbon while RNA has an OH at the 2' Carbon on pentose.

2. DNA has a methyl group associated with the pyramidine Thymine which contributes to it's stability, RNA's uracil does NOT have a methyl group
Besides building nucleic acids, what is another function of nucleotides?
nucleotides carry energy in the cell by phosphodiester bonds

The more Phosphates, the more energy it has
Describe Coenzyme A
IT has adenosine linked to phosphate

Phosphoadenosine diphospate

It functions in acyl group transfers
Describe NAD+ and FAD
carry electrons.

Both contain nucleoside adenine.

Nicotinamide adenine dinuc.

Flavin adenine dinucleotide
Importance of Nuclotides as regulatory molecules
cAMP
CMP
ppGpp

good as second messangers
cAMP
adenosine 3'-5' monophosphate

has a phosphate group linked between the 5' to the 3' OH making a cyclic ring. This singans ATP depletion
ppGpp
has 2 phosphates on the 5' end and 2 phosphates on the 3' end. Guanine is the nucleoside.

signals aa starvation in bacteria which leads to gene induction
polymer of nucleotides
Nucleic acids
How are nucleotides linked together to make nuclic acid?
phosphate group is on 5' end
hydroxyl group is on 3' end
the nucleotides are linked by phosphodiester bond due to condensation rxn between phosphate and hydroxyl groups
why does A=T and C=G
bc DNA is double stranded and A binds to T and C binds to G
Describe DNAs distinctive base compositons
base composition varies from species to species. Each species has a certain percent base pair.

Base compostion does NOT vary from tissue to tissue wi the same species. It remains constant with age, nutrition and env. throughout life.
What defines DNA base composition?
number of As,Gs,Ts,and Cs
Chargaff rules (1940)
A=T and G=C
so A+G = C+T
where are bases in DNA double helix?
in center of Double Helix

The outer strands are composed of the sugar and phosphate diester bonds
why are the major and minor groove important in the DNA double helix?
bc DNA binding proteins, regulatory proteins are very specific in whether they bind to major or minor grooves
3 types of DNA
B DNA
A DNA
Z DNA
B DNA
Nomal DNA found in Cell

Right HANDed
A DNA
Dehydrated form of DNA

Right handed
Z DNA
produced in lab, may exist in cell

Left handed
General structure of DNA
Composed of two individual strands that are antiparralel to each other and bases are complementary (A=T and C=G)
antiparallel
one strand runs 3' to 5' and the other strand runs 5' to 3'
in double helix how are the two strands bound together?
By Hydrogen bonds between bases
Number of H bonds between C and G

Number of H bonds between A and T
C and G are held together by 3 H bonds

A and T are held together by 2 H bonds

Therefore, C=G is bound tighter
distance betwee bases with H bond
3A
describe how base stacking contributes to DNA helix stability
base pairs tack on top of one another due to hydrophobic forces bc bases are hydrophobic.

Pretty cool...free nucleotides in solution, the bases will stack spontaneously forming a helix in solution
what contributes to the stability of DNA double helix?
DNA base stacking
describe DNA semi conservative replication
allows genetic infor to be accurately replicated

after replication you have a hybrid molecule consisting of one parental strand and one newly synthesized strand.
Why is it a DNA world?
bc DNA is more stable in aqueous solution than RNA is which is good considering we are made of aqueous sln.

RNA in aqueous sln is bad bc if a hydroxyl group comes in it attacks the 2'OH and cause a cyclic intermediate between the 2' and 3' groups and the phosphodiester bond breaks seperating the molecule. This rxn is catalyzed by RNAase
Structural variation in DNA
palindrome sequence and mirror repeat can cause harpins and cruciforms.

Free rotation between sugar and base and can lead to cis or trans structures (both are stable tho)

H bonds can lead to 3 or 4 stranded DNA
What causes hairpins and cruciforms in DNA?
regions with symmetry facilitate these alternate structures
H DNA
Triplex from a Duplex

due to a sequence, the strands seperate and one strand loops out and the unbound strand Hydrogen bonds to another DNA strand

This occurs spontaneously in Bacteria cells
What are some secondary structures that RNA molecules form?
Bulge
Internal loop
hairpin

this allows lots of variation in tRNA and rRNA
how is tRNA 3D
although RNA is single stranded, the RNA folds up and becomes a 3D structure with alternate base pairs that consist of minor nucleotides too!
Can DNA from diff species form hybrids?
yeah if they are closely related
What happens if you heat DNA?

Recool?
If you heat DNA you add enough energy to break the H bonds and the Double helix will seperate (denature)

When you cool the DNA the bases come back together bc of Watson Crick base pairing
What is important about a species Tm of DNA?
Tm is the point where the DNA is 50% denatured.

DNA from different species has different 50% denaturation points (Tm) It has to do with the base composition. More G=C bonds, the DNA is more stable so it will take longer to denature and it will have a higher Tm compared to A=T rich DNA
experiment to see how closely 2 organisms are related
Denature DNA

Mix the different DNA

Cool (annealing)

If the DNA strands are closely related H bonding will occur where v. similiar genes are present. Use electron micrograph to examine where the 2 molecules are bound (areas of similiarilty) and where the 2 DNAs differ there will be divergence
Gene
segment of DNA molecule that contains the information required for the sytheiss of a functional biological product whether protein or RNA
Only function of DNA
storage and transmission of genetic information
Central Dogma
DNA is master blue print that is transcrbed to RNA. RNA is then translated to protein
Avery, McLeod, and McCarty 1944
Greates experiemt that did NOT get a nobel prize. Showed that the transfer of genes for the bacterial capsule was via DNA. Show that DNA was transforming substance
Who concluded that genes were made of DNA?
Avery, McLeod, McCarty 1944
Hershey Chase Experiment
1952
Confirmed that DNA was the genetic material.

they used bacteriophages with either radiolabeled DNA or radiolabeled proteins.

Only the radioactive DNA was found injected into host cell proving DNA is genetic material
What has a broader range of function, DNA or RNA?
RNA

it can be rRNA, tRNA, mRNA

RNA can carry out catalytic functions with ribozymes
how are genes organized?
Why?
Genes are organized into chromosomes

Chromosomes allow organization of genes into larger funcitonal units

Chromosomes ensure that all genes are equally replicated so that no genes are left behind
2 ways that bacterial genes and eukaryotic genes differ
1. Bacterial genes are usually polycistronic. Eukaryotic genes are monocystronic

2.Eukaryotic genes have exons and introns
monocistronic mRNA
mRNA that can be translated into only one proteins.

one gene is transcribed by only one promotor
polycistronic mRNA
an mRNA with more than two genes that can be translated into proteins

(Bacteria do this!)so many mRNA can be transcribed at one time
describe exons and introns
only in eukaryotes

genes are split up into exons which are the expressed units and introns which are removed. Intron are intervening sequences that are split up exons so introns must be removed to get final mRNA
DNA replication overview
DNA replication is semi conservative

Replication begins at orgin and proceeds bidirectionally

DNA synthesis proceeds in a 5'->3' direction and is semi discontinuous
what does semi conservative mean?
DNA is semiconservative which means that newly replicated DNA molecule contains one old strand and one newly synthesized strand
How does DNA replicate bidirectionally?
sTARTS at an orgin and replication goes from orgin in both directions
enzyme family that synthesizes DNA
DNA polymerase catalyze the rxn:

dNMP(n) + DNTP->dNMP(n+1)+PPi

basically adds one nucleotide residue to DNA
in what direction does DNA polymerase synthesize DNA?
5' --> 3'
What do DNA polymerases require?
1. Template strand
2. A short primer to initiate synthesis

What happens is that a RNA polymerase (primase) recognizes a certain DNA template and makes a RNA primer that the DNA polymerase can bind to and start synthesizing DNA
Do RNA polymerases need a primer?
NO! they actually make the primer for DNA polymerase
describe elongation of DNA chain
incoming deoxynucleotide triphosphate comes in and bind to growing strand and inorganic phosphate is released, now a new base is added!

The complementary strand is made from the template strand
Compare DNA polymerase I and III
DNA polymerase I has:
1 subunit
3'->5' exonuclease
5'->3' exonuclease
so it can cleave from either end

DNA polymerase III has:
over ten subunits
3'->5' exonuclease but does NOT have a 5'->3' exonuclease
Main polymerase used to replicate DNA/chromosomes
Poly III
Is DNA accurate? WHY?
DNA replication is v. accurate. DNA polymerase makes one mistake in 10000000
bc.....
1. 3'->5' proofreading exonuclease

2. specific DNA repair systems recognize errors and mismatches
Since it is possible for G to H bond to A and C to H bond to A, why doesnt it happen?
bc DNA polymerase I does not accept this!
enzymes that degrade DNA
nucleases
endonucleases
attack and degrade DNA at specific internal site. They usually recognize a specific DNA sequence. Endonucleases are known as Restriction enzymes
What do MOST DNA polymerases have to help correct mistakes?
3' -> 5' exonucleases
2 active regions of DNA polymerase
1. DNA polymerase active site

2. 3' -> 5'proofreading exonuclease active site
give an example of how polymerase and proofreading exonuclease work together to ensure DNA fidelity
say a C comes in and Hydrogen bonds with an A, the polymerase recogizes the mistake and backs up and uses a 3' ->5' exonuclease to cleave off the wrong base and try again
What is RNA primer removed from DNA?
DNA polymerase I binds to the gap between RNA and DNA, it proceeds along the strand and removes the RNA primer with 5' -> 3' exonuclease and synthesizes and new DNA strand simultaneously
Problems that must be overcome for DNA replication
1. supercoiling

2. Primer for intiation of DNA polymerase

3. Leading vs. lagging strand
What enzymes allow DNA polymerase to get into supercoiled DNA? How?
DNA is v. supercoiled so it can fit into the nucleus. First DNA B (helicase) seperates the two strands and pulls them apart.

DNA gyrase relieves the topological stress caused by supercoiling to relax the DNA
Describe How DNA replicates lagging strand
So DNA polymerase finds template that is 3' to 5' binds a synthesizes DNA in a 5' to 3' direction on leading strand w/ no problem, it is synthesized continuously but with lagging strand the DNA poly cant synthesize from 3' to 5' so primase lays down some RNA primers so DNA poly can synthsze short fragments of DNA called Okazaki fragment and gaps are between them so now DNA poly I comes in and removes RNA and puts down DNA to close gaps
principle DNA poly for replication
DNA polymerase III
steps of DNA replication
initiation

elongation

termination
describe DNA replication initiation
Occurs at a specific DNA site called an orgin which is a certain sequence. At orgin, DNA A protein binds. DNA A proteins is an ATP dependent protein that forms a multiple subunit and pulls apart strands so that the DNA helicase can come in and bind and unwind the strands so that DNA polymerase can assemble at this place
Single stranded binding proteins
part of RNA polymerase complex these proteins coat the DNA and prevent the strands from going back together
Final step in DNA lagging strand synthesis
After DNA polymerase III replicates the chromosome, DNA polymerase I comes in and uses nick translation to remove the RNA primer but there are still gaps present so Ligase uses ATP and seals gaps to get final replicated DNA strand
How does DNA ligase work?
DNA ligase is an enzyme that loads itself with ATP and donates the phosphate from the ATP to the gaps of DNA and AMP leaves. The phosphate seals up the gaps in DNA so you have a newly made 5' to 3' DNA strand wo gaps
describe termination of replicatoin in Bacteria like E. coli
termination signals are v. important in bacteria bc the DNA is circular. So there are DNA sequences on each side of the orgin on the bacterial DNA to trap the DNA polymerase, trapping the replication fork
After duplication, the chromosomes get tangles and mixed up, how do they get fixed?
sometimes after many rounds of replication, the DNA gets mixed up so DNA topoisomeraes nick and break and seperate the tangeled DNA to get the strands to seperate
Describe how DNA pol II is coordinated for DNA repication
DNA polymerase stays still and the DNa is pulled through it.

At the orgin of repication you have 2 DNA pol III complexes one goes 3' to 5' and the other is 5' to 3' each synthesize a leading and lagging strand
describe a breif summary of the difference in eukaryotic DNA replication
Eukaryotic DNA has linear chromosomes that are way bigger than prokaryotic DNA so there has to be many sites of replicaton. DNA gamma in eukaryotes is similiar to DNA polyIII in bacteria. The termination of repication in eukaryotes occurs at telomeres
3 types of antimicrobials that target DNA metabolism
1. those that interfere w/ nucleotide synthesis

2. Those that inhibit DNA polymerase

3. Those that inhibit RNA replication
examples of antimicrobial drugs that interfere w/ nucleotide syntheisis of bacteria
Flucytosine Antifungal agent

antivaral agent

AZT Anti HIV (prevents reverse transcriptase)
Example of an antimicrbial that inhibits DNA directed DNA polymerase
rifamycin an antibiotic
Why can we have antimicrobials that target DNA metabolism?
bc DNA in bacteria is different than eukaryotic DNA
How does acyclovir the anti Herpes drug work?
since herpes is virus that brings in its own DNA into the cell, Acyclovir is a nucleotide analouge that is a terminator that stops the replication of viral DNA
How are DNA sequencing and actual repication in the cell similiar and different?
both use DNA template strand, dNTPs, primer, and DNA polymerases but only DNA sequencing in lab uses dideoxnucleotides as terminators
purpose of dideoxynucleotides aka chain terminators
they look like dNTPs but they lack a 3' OH so when you put ddNTP into a growing DNA strand it causes termination of DNA synthesis since there is no 3' OH to link to 5' Phosphate group
How to do DNA sequencing
each rxn should contain all four dNTPs and a ddNTP or else DNA synthesis would not proceed past the first ddNTP so certain bases can be sequnced
described automated DNA sequnecing
add all four dNTPs tagged with fl- with four ddNTPs

denature

load into cap get and apply and electrical current

using a laxer it can detect each label fl- dNTP based on different color to sequence them
3 main mistakes that occur to nuclotides and nucliec acids that cause mutation
1. mistakes in replication. When DNA polymerase makes a mistake which only happens in one in 10 millionbases

2. Spontaneous sequence changes which occurs by chemical nature of nucleotides due to tautomerization

3. The action of chemical or environmental mutagen that damages the DNA
Mutation
heritable changes in the genome. A change that occurs in the DNA sequence
What are the accumulation of mutations linked to?
genetic diseases carcinogenesis, and aging
2 groups of mutations
1. somatic mutations
2. germline mutations
Mutations arise in a _______ and spread by either ________ or _____________
mutations arise in a single cell and spread by either cell diviesion or reproduction
Somatic mutations
somatic changes are mutatuion that can occur in any cell in the body except for eggs or sperm. These changes can be maintained in the body by cell division but CANNOT be passed on to offspring

ie: Cancer cells
Germline mutations
mutation that have occured in a zygote (egg or sperm) that can be passed on to progeny. When passed on by inheritence EVERY cell in the descendent will have the mutation
2 step process of fixed mutations
1. damage to DNA in one strand causes mismatch of Watson base pair. this is considered "premutagenic"

2. After DNA replication, now mutation is in both strands. Once mutatoin is replicated, it is fixed in the genome
What is the ames Test?
the Ames Test is used to detect mutagenic substances and chemicals. Uses strain of bacteria that reverts back to histidine synthesis when exposed to mutagens
Types of DNA repair systems in Ecoli
MMR
Base excision repair
nucleotide excision repair
Direct repair
main post replication repair system
mis match repair
Type of repair that looks for abnormal bases incorporated into DNA
Base excision repair
major post replication DNA repair system in GRAM neg bacteria
Methyl directed mismatch repair

Humans have mismatch repair but it is not with methylation
How does mismatch repair use methylation?
Uses DNA methylation for strand discrimination between the methylated template (old) strand is distinguished from the unmethylated (new) strand.
Corrects the error in the unmethylated strand
Describe cells that have defective MMR. Why is this so important?
Cells that are defective for MMR show mutation rates that are 100-1000 times higher than normal; they are said to have a mutator phenotype.

this is seen in some pathogenic bacteria which is really bad bc they are often antibiotic resistant
At what point can MMR work?
only with hemimethylated DNA. Right after replication, when one strand is methylated and the other is not yet
Describe MMR 1 and MMR 2
MMR1 finds mistake in DNA so MutS and MutI bind, later MutH joins complex, DNA is pulled through complex until comes to bulge where the bases dont match up and MutH puts nick in unmethylated strand.

MMR2 use exonuclease to degrade the DNA mistake and then DNA polymerase III comes in and fixes it
frequency of MMR defect in humans
1/200 this causes increase in cancer frequency. Especially colon cancer
Describe Base excision repair
BER looks for specific altered residues

First, a damaged or inappropriate base is removed from its sugar linkage by a glycosylase enzyme which cut the base-sugar bond

Second, the AP endonuclease then removes the remainder of the damaged nucleotide causing a gap in the DNA

Third, DNA polymerase I does nick translation to repair base and ligase seals it up with Phosphate
major repair mechanisms in mammals
nuclotide excision repair
describe nucleotide excision repair
Recognizes DNA damage which is "bulky" and creates a block to DNA replication and transcription

first Cleaves DNA strand containing the damage by endonucleases cutting on both sides of damage

followed by exonuclease removal of a short segment containing the damaged region (~30 bases in mammals and 13bases in e.coli)

DNA polymerase I (E) fills in the gap and DNA ligase seals it up
xeroderma pigmetosum
Humans with Autosomal recessive disease of defective nucliotide excision repair.

Pts are sunlight sensitive and have hi risk of cancer on sun exposed areas of body
Photoreactivation in bacteria
Uses DNA photolyase to split pyrimidine dimers in presence of white light
problem and repair of mehylation G
Methylation of G can lead to mutation bc methylated Gs can bind to T.

Repair methlated guanine with methyltransferase to remove the methyl group
What repairs Alkalated bases?
Alk B
Genetic Recombination
Rearrangement of genetic info wi DNA
3 main mechanisms of recombination
homologous recomination

site specific recombination

DNA transposition
Where does homologous recombination occur?
between two v. similiar DNA sequences

occurs in both eukaryotes and prokaryotes
since homologous recombination occur between two identical DNA sequences what is needed?
MMR for ensuring DNA specificity. WO MMR the recombination can occur between any DNA sequences and this is not good at all
Purpose of Homologous recombination in bacteria
repair

genetic diversity
Purpose of Homologous recombination in eukaryotes
repair
replication
cell division
diversity
two key enzymes involved in homologous recombination
1. RecA is the key enzymer (it is a SS binding enzyme so depends on RecBCD).Rec A pairs the 2 DNAs, forms the holliday intermediates, and allows branch migration

2. RecBCD enzyme complex. generates the SS substrate for RecA to bind to, it helps initiate homologous recombination. It hlps helicase and nuclease activity and it recognizes chi site
What does Rec BCD enzyme complex recognize?
Chi sites
5'GCTGGTGG3'
How does RecBCD work?
RecBCD binds to double stranded DNA and in an ATP dependent fashion, uses nucleases and helicases to unwind and degrade DNA until it gets to a chi site. At the chi site, it inhibits the nuclease on one strand only and the other strand continues to be degraded making a SS end which is ideal for initiating homologous recombination
What two main things does RecA cause?
Strand displacement and strand exchange
how does RecA work?
RecA protein coats SS DNA and mediates a strand invaseion to make homologous DNA duplex. Using ATP, RecA unwinds the 3 stranded intermediate. Using another ATP, it causes branch migration to remove/displace one strand of the DNA
What can rescue stalled replication due to lesion in DNA?
homologous recombination allows DNA to get replicated
Homologous recombination is used for multiple tasks in the cell:
DNA Repair
Restarting of stalled replication forks
Generation of genetic diversity during meiosis
DNA Replication
Generation of antibody diversity
Difference in homologous recombination and site specific recombination
site specific occurs at a specific DNA sequence that is shared by two molecules where homologous recombination can occur at any shared sequence

Site specific recombination does not require RecA
Properties of Site specific recombination
Recombination occurs at a specific DNA sequence that is shared by two molecules (homologous recombination can occur at any shared sequence)

Can mediate DNA integration & excision as well as DNA inversion

Mediated by an enzyme (recombinase) that recognizes that specific sequence
Recombinase often called an Integrase

Does not require RecA

Used by bacteriophages (phages, bacterial viruses) to integrate their DNA into the bacterial DNA
evidence of site specific recombination
always a duplicated target sequence where it took place
2 consequences of integration due to site specific recombination
1. INVERSION- if orientation of the duplication is inverted upon site specific recombination, it causes inversion of the original sequence

2. DELETION AND INSERTION
lining up of direct repeats with site specific recomb. it spits out DNA causing chromosomal deletions.
How do bacteriophages get in and out of chromosomes?
Using site specific recombination insertion and deletions.

this allows phages to integrate into bacterial genome and bring bad genes in w/ them which increases virulence
Jumping genes
transposons
Transposons and mobile DNA elements
Mobile DNA elements that are often associated with antibiotic resistance
Transposons encode the protein that mediates transposition: transposase enzyme
Target DNA sequence is duplicated when the transposon inserts itself
Two main types of transposition
Simple transposition
“cut and paste” method
Leaves a double-strand break in donor strand that must be repaired

Replicative transposition
Proceeds via formation of a cointegrate intermediate
Donor & recipient both end up with copies of the transposon. It replicates itself so that both the original and new have transposen
Transposen enzyme used to move to another chromosome
Transposase
How do transposons work?
Transposease makes staggered cuts in target sequence and transposon is inserted in at site of cuts. Replication fills in the gaps
Hallmark of traspositon
Duplication
type of recombination that does AB genes
site specific recombination
How do Ab assemble and be diversed?
RECOMBINATION
Sometimes after recmobinational repair of circular bacterial chromosome, a dimer forms. If dimer is uncorrected the replicated molecule cant segregat into the daughter cell upon division. What fixes this?
XerCD a special site specific system for Ecoli seperates the chromosomes.
describe transposon segment
have terminal repeats on each end
other functions of nucleotides
carry energy into cell with phosphodiester bond

adenosine nucleotides act as cofactors

electron carriers (NAD and FAD)

regulatory molecules (cAMP,cGMP, ppGpp)
What are the 5 main steps of protein synthesis?
1) Amino acid activation
2) Initiation
3) Elongation
4) Termination,
5) Polypeptide modification (primarily folding and processing
which stages of translation occur on the ribosome?
initiation
elongation and termination
what toes translation require?
mRNA in cytosol

3IF
3EF
3RF (in bacteria)
How are IF2 and EF Tu similiar and different?
they both bind to tRNA with GTP

but the differ bc IF2 is bound to fmet-tRNA containing GTP so IF2 recogizes only one class of tRNA which is the initiation tRNA

EF Tu(GTP) bind to all other aa tRNAs and Met-tRNA
What are the functions of the three prokaryotic initiation factors?
IF1 and IF3 bind to the small ribosome subunit. IF1 blocks a second (acceptor) site used for elongation and IF3 prevents the large subunit from attaching until after f met tRNA binds. IF2 contains GTP and binds to f met tRNA
describe initiation of protein synthesis in bacteria (how initiation complex is made)
IF1 is bound to A site to block it and IF3 binds to 30S so that the large subunit cant bind

30S subunit binds to mRNA at the Shine Dalgarno sequence.

IF2 w/GTP binds fmet tRNA anticodon to codon AUG at P site

IF3 falls aways so 50S can attach (makes 70S complex)

GTPase on 50S hydrolyzes GTP on IF2 so IF2 falls away and so does IF1 so A site now ready
Describe Shine Dalgarno sequence
upstream for AUG composed of series of purines. This is where mRNA binds to the small subunit. SD permits polycistronic mRNA translation

only in bacterial mRNA
Translation Initiation in eukaryotes differs from that in prokaryotes in 3 major ways. What are they?
a) The 40S subunit binds the mRNA cap and tail
b) The 40S subunit slides along the mRNA to the AUG codon using a helicase (no Shine Dalgarno nucleotide sequence)
c) More initiating factors than in prokaryotes
requirements to initiate translation in prokaryotes
1. mRNA with Shine delgarno sequence, AUG, and f met tRNA

2. 30S ribosome with IF1 and IF3 bound

3. f met tRNA met with IF2 and GTP

4. 50 S

5. Mg cofactor for translation

this whole thing is the 70S initation complex
Indicate the 4 major steps of elongation and what the respective major cofactors are?
1) Proof reading
2) Delivery
3) Peptide bond formation
4) Translocation.
EF TS EF Tu and GTP are associated with proofreading and delivery.
Peptidyl synthetase, EF G and GTP are associated with peptide bond formation and translocation
describe proofreading and delivery part of protein elongation
1. EF-Tu(GTP) bound to aa tRNA comes in to A site for long enough time for GTPase of 50S to hydrolyze the GTP-EFTu (proofreading) now it is GDP-EFTu the GDP is replaced by EFTs. Then Ts is replaced by another GTP which can bind to another aa tRNA and start cycle again
describe peptide bond formation and translocation
peptide synthetase in large subunit causes amino group from A site to attack the ester bond in Psite and the tRNA in the P site transfers to the E site so aa in A site attach to aa on P site but they are in twisted state so EFG-GDP binds to stabilize twisted state. GDP is exchanged for GTP. the EFG-GTP causes transition to P site so now A site is open for incoming codon
What is unique about the codon for transcription termination?
Signaled by termination codons that bind protein release factors instead of an amino acyl tRNA
describe release of peptide
RF1 or RF2 binds and exchanges the GDP on RF3 to GTP. RF3(GTP) by the ribosomal GTPase causees dissciation
Calculate the ATP equivalents (used as GTP or ATP) needed to synthesize a polypeptide during translation
Two ATPs are required to activate (make aa-tRNA), 1 GTP/aa for initiation or delivery (IF 2 and EF Tu) and 1 GTP for translocation or termination. Total is 4 ATP or equivalents for every amino acid present in the polypeptide
What are the 5 main steps of protein synthesis?
1) Amino acid activation
2) Initiation
3) Elongation
4) Termination,
5) Polypeptide modification (primarily folding and processing
which stages of translation occur on the ribosome?
initiation
elongation and termination
what toes translation require?
mRNA in cytosol

3IF
3EF
3RF (in bacteria)
How are IF2 and EF Tu similiar and different?
they both bind to tRNA with GTP

but the differ bc IF2 is bound to fmet-tRNA containing GTP so IF2 recogizes only one class of tRNA which is the initiation tRNA

EF Tu(GTP) bind to all other aa tRNAs and Met-tRNA
What are the functions of the three prokaryotic initiation factors?
IF1 and IF3 bind to the small ribosome subunit. IF1 blocks a second (acceptor) site used for elongation and IF3 prevents the large subunit from attaching until after f met tRNA binds. IF2 contains GTP and binds to f met tRNA
describe initiation of protein synthesis in bacteria (how initiation complex is made)
IF1 is bound to A site to block it and IF3 binds to 30S so that the large subunit cant bind

30S subunit binds to mRNA at the Shine Dalgarno sequence.

IF2 w/GTP binds fmet tRNA anticodon to codon AUG at P site

IF3 falls aways so 50S can attach (makes 70S complex)

GTPase on 50S hydrolyzes GTP on IF2 so IF2 falls away and so does IF1 so A site now ready
Describe Shine Dalgarno sequence
upstream for AUG composed of series of purines. This is where mRNA binds to the small subunit. SD permits polycistronic mRNA translation

only in bacterial mRNA
Translation Initiation in eukaryotes differs from that in prokaryotes in 3 major ways. What are they?
a) The 40S subunit binds the mRNA cap and tail
b) The 40S subunit slides along the mRNA to the AUG codon using a helicase (no Shine Dalgarno nucleotide sequence)
c) More initiating factors than in prokaryotes
requirements to initiate translation in prokaryotes
1. mRNA with Shine delgarno sequence, AUG, and f met tRNA

2. 30S ribosome with IF1 and IF3 bound

3. f met tRNA met with IF2 and GTP

4. 50 S

5. Mg cofactor for translation

this whole thing is the 70S initation complex
Indicate the 4 major steps of elongation and what the respective major cofactors are?
1) Proof reading
2) Delivery
3) Peptide bond formation
4) Translocation.
EF TS EF Tu and GTP are associated with proofreading and delivery.
Peptidyl synthetase, EF G and GTP are associated with peptide bond formation and translocation
describe proofreading and delivery part of protein elongation
1. EF-Tu(GTP) bound to aa tRNA comes in to A site for long enough time for GTPase of 50S to hydrolyze the GTP-EFTu (proofreading) now it is GDP-EFTu the GDP is replaced by EFTs. Then Ts is replaced by another GTP which can bind to another aa tRNA and start cycle again
describe peptide bond formation and translocation
peptide synthetase in large subunit causes amino group from A site to attack the ester bond in Psite and the tRNA in the P site transfers to the E site so aa in A site attach to aa on P site but they are in twisted state so EFG-GDP binds to stabilize twisted state. GDP is exchanged for GTP. the EFG-GTP causes transition to P site so now A site is open for incoming codon
What is unique about the codon for transcription termination?
Signaled by termination codons that bind protein release factors instead of an amino acyl tRNA
describe release of peptide
RF1 or RF2 binds and exchanges the GDP on RF3 to GTP. RF3(GTP) by the ribosomal GTPase causees dissciation
Calculate the ATP equivalents (used as GTP or ATP) needed to synthesize a polypeptide during translation
Two ATPs are required to activate (make aa-tRNA), 1 GTP/aa for initiation or delivery (IF 2 and EF Tu) and 1 GTP for translocation or termination. Total is 4 ATP or equivalents for every amino acid present in the polypeptide