term 1 pos

basic structure of chromosomes-

• centromere at centre

• telomeres at ends- protective, repetitive sequence of DNA

• there are replication origins

• genes spread around

structure of a eukaryotic gene

• promotor region

• exons and introns

• TATA box in promotor- initiation of transcription

• transcription initiation site

• translation initiaton site in exon 1 (ATG)

• translation termination site

• transcription termination site

what is the central dogma

DNA to RNA to protein

(transcription, splicing, export, translation, folding)

what are RNA polymerases and what are the 3 types

• protein complexes made up of 8-14 subunits

• Pol 1 - ribosomal RNA

• Pol 2- mRNA for translation into protein

• Pol 3- small RNA (tRNA)

transcription initiation

• transcription factor binds to eukaryotic promotor

• formation of preinitation complex

• RNA pOL 2 begins elongation

what do transcription factors bind to

• gene specific transcription factors bind to regulatory sequences of the gene

gene definition

A locus (or region) of DNA, which is made up of nucleotides and is the molecular unit of heredity

chromosome definition

A thread-like structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes.

genome definition

The complete set of genes or genetic material present in a cell or organism

initiation

• general transcription factors bind , eg TBP to TATA box

• pre initiation complex formed

• rna polymerase holoenzyme binds to the promotor region

where do transcription factors bind to?

regulatory sequences

what is a transcription factor homodimer

2 protein monomers that bind together to regulate gene expression

trans activating domain

regions of transcription factors which bind to coactivator complexes to activate transcription

helix turn helix motif

• two alpha helices joined by a short strand of amino acid

• recognition and binding of DNA is done by the 2 helices, one at n terminus and one at c terminus

• c terminal binds to major groove, n terminal helps to position the complex

other dna binding motifs

• zinc finger

• leucine zipper

• helix loop helix

elongation

• what does rna polymerase do

• what helps maintain the correct length

• what does the zipper do

• RNA polymerase travels along from the 5’ to 3’ end (upstream to downstream)

• rudder in the upstream end of the RNA-DNA hybrid helps maintain the correct length, etc

• the zipper is another protein loop within RNA polymerase and maintains boundaries of the transcription bubble especially at the downstream end

what are topoisomerases

regulate dna overwinding or underwinding

5’ cap addition

• 7 methyl guanosine triphosphate cap

• this is a modified guanine cap to the 5’ end of mRNA

• protection from degradation

• initiation of protein synthesis

3’ polyadenylation

• example of the sequence

• what recognises it

• what happens then to the mRNA

• what then happens at the 3 end

• what binds

• why does this happen

• sequences of the pre mRNA such as the AAUAAA sequence are recognised by the cleavage and polyadenylation specifity factor (CPSF)

• then an endonuclease cleaves mRNA at specific site

• enzyme poly-a-polymerase joins multiple adenine nucleotides to the 3’ end

• poly a binding proteins bind to the newly formed tail

• provides stability and facilitates exit of mRNA from the nucleus

xm2

nuclease that degrades remaining nacsent transcript and leads to termination of transcription

splicing

• introns always begin with the residues GU and end with AG and an A somewhere towards the middle

• phosphodiester bond is attacked between GU and A

• 3 prime end attacks phosphodiester bond between A and AG joining exons and removing the introns

how can proteins be altered for a specific function?

alternative splicing

what is the genetic code

• 20 amino acids

• 4 diff nucleotides

• if code is x nucleotides , number of possiblity is 4 to the power of x

• codons

charging of tRNAs with amino acids

• what does amino acyl tRNA synthetase do

• what does it then attach and what is released as a result

• what is created

• what does the enzyme bind this to

• what then happens and what kind of bond is formed

• what molecule is released

• amino acyl tRNA synthetase binds an amino acid and ATP

• the enzyme catalyses a reaction that attaches AMP to amino acid releasing pyrophosphate 

• this creates a high energy aminoacyl-adenylate intermediate

• The same enzyme, aminoacyl-tRNA synthetase, binds the activated aminoacyl-adenylate and its corresponding tRNA molecule.

• amino acid is transferred from AMP to the 3 end of tRNA which forms an ester bond

• AMP is released, as the amino acid is now covalently linked to the tRNA, creating a "charged" aminoacyl-tRNA. 

what are the site of the ribosome

• p site holds trna with growing polypeptide chain attached

• e site holdsthe trna which will exit

• a site holds aminoacyl trna

elongation cycle

• what is it

• what is delivered to the A site by what. what does this consume

• what catalyses formation of a peptide bond

• what happens and what 2 things does this need

• process of adding amino acids to a polypeptide chain

• aminoacyl trna is delivered to the A site. elongation factor escorts the trna and binds it to the ribosome consuming GTP (to GDP)

• peptidyl transferase catalyses formation of peptide bond

• ribosome shifts one codon along in the 3 direction. facilitated by elongation factor 2, this requires GTP.This action moves the tRNAs: the now uncharged tRNA from the P-site moves to the E-site, and the tRNA carrying the polypeptide chain moves from the A-site to the P-site, freeing up the A-site for the next cycle. 

translation initiation

• what do the eukaryotic initation factors bind to

• what does initiator tRNA carry and what does this do

• what does the small ribosomal subunit do

• what happens to the GTP on the tRNA and what does this cause and allow

• what sits on the p site

• eukaryotic initiation factors bound on the 5’ end join to the (poly a binding protein) bound on the poly a tail of the 3’ end

• initiator tRNA carries methionine, GTP and initiation factor 2, and the 40s ribosome both recognise the initiation factors bound to the 5 and 3 ends. They all interact

• small ribosomal subunit finds the start codon, using protein initiation factors. but some are easier to find than others (purine 3 residues upstream of A -Kozak sequence) 40s subunits slides down

• GTPase activity converts the GTP on the tRNA to GDP 

• causes conformational change, and GDP is release

• this allows the large ribosomal subunit to bind

• methionine sites on the p site of the ribosomal complex

• ready to begin elongation

termination

• stop codon do not have a corresponding trna to recognise

• instead when a ribosome reaches the stop codon the a site accepts a protein called release factor instead of tnra

• release factor hydrolyses the bond between trna in the p site and the amino acid in the polypeptide chain, freeing it

• two ribosomal subunits and other structures dissociate

antibiotics targeting translation

• chloramphenicol

• erythryomycin

• tetracycline

• streptomycin

• chloramphenicol binds to 50s rrna inhibiting formation of a peptide bond

• erythromycin binds to 50s of rrna and prevents movement along mrna

• tetracycline inhibits trna anticodon reading of mrna codon

• streptomycin changes shape of 30s rrna and causes mrna to be read incorrectly

what is meant by a protein structure hierarchy

-primary, secondary, tertiary, quaternary(eg 2 beta and 2 alpha globin polypeptides with a heme group)

• what type of amino acids are mammalian proteins

• how many amino acids

• what is the peptide bond classified by

• how many amino acids makes a protein

• shape of peptide bond

• mammalian proteins are alpha amino acids

• 20 amino acids

• carboxyl group reacts with amino group to form a peptide bond and lose a water molecule

• catalysed by peptidyl transferase (28s ribozyme-ribonucleic acid enzymes)

• within a polypeptide chain, individual amino acids = residues

• when >50 amino acids are in a polypeptide is a protein

• peptide bond is planar (partial double bond between carbonyl O and N)

bonding determining protein structure

• covalent primary

• hydrogen secondary

• hydrophobic+VDW tertiary

• electrostatic within tertiary helps maintain

• VDW/ electrostatic quaternary

alpha helix

• what runs parallel to the helix axis

• what winds around the axis

• what does htis lead to

• how many amino acid residues per complete turn

beta sheets

• what orientation do H bonds run to the chain direction

• alpha helix- H bonds run parallel to helix axis. alpha carbon backbone winds around an axis so that each carbonyl O atom is H bonded to each amino N of the amino acid located 4 residues closer to C terminus

• The standard alpha helix has approximately 3.6 amino acid residues per complete turn

• beta sheets- H bonds run perpendicular to chain direction

• parallel and antiparallel

types of r groups

nonpolar, polar, electrically charged

cysteine

can form disulphide bonds as it has an SH group

examples of tertiary proteins

-catalase

-triose phosphate isomerase

-actin

tertiary domain

the smallest stable unit of a tertiary structure. a domain is defined as that region of a polypeptide chain that can fold into an autonomous stable tertiary structure

domain shuffling- when domains have been switched around between proteins through evolution

haemoglobin- quaternary potein

• what are the two states

• how many units

1. haemoglobin- needs to deliver oxygen tissues. needs high enough affinity to pick up oxygen but low enough to release it.

2. t state- oxygen unbound

3. r state- oxygen bound. so that the 3 other units have higher affinity

4. small changes in oxygen concentration can dramatically affect binding to haemoglobin giving a sigmoidal curve of oxygen binding

collagen

• what type of protein

• what is the general structure

• extracellular fibrous protein but not an alpha helix

• 3 helical chains that wind around a central axis

• general structure is Gly-X-Y where X can be any amino acid especially proline lysine or hydroxyproline

• Glycine is small so glycines from each chain fit at the centre of each helix

• H bonds between the chains

functions of chromatin

• package and condense dna

• prevent dna damage

• control dna expression

what are nucelosomes

• how many base pairs

• what is the core comprised of

• what are the cores separated by

• what brings nucleosomes together and what does this do

• what do multiple stacked rings form

• what is formed when they are coiled further

• ‘beads on a string’

• around 146 base pairs of dna wrapped around 1.7 times around a core

• core is comprised of an octamer of histone proteins

• nucleosome core proteins are separated by linker proteins

• histone H1 brings nucelosomes together to condense chromatin further

• multiple stacked rings form the solenoid fibre/30nm fibres

• these are coiled further to form chromosomes

pcr

• heat to 94 to denature (separate) dna strands

• cool to 50-60 to allow primers to anneal

• 72 to allow elongation of new strands using taq polymerase

• so at the end of the first cycle 2 new strands

• begin cycle two by denaturing and annealing again

• elongation generates strands of different sizes depending on the template strands

sanger sequencing

• mix small amount dideoxy with deoxy nucleotides

• cycle through multiple rounds of pcr

• each ddNTP contains fluorescent tags

• separate by gel electrophoresis

• detect by laser

• (chain synthesis terminates when ddNTP is incorporated)

technical applications of PCR that could be used to an advantage in veterinary medicine

• VNTR

• RAPD

• AFLP

• they are all used to determine breed, pedigree and/or parentage

what components are essential for basic PCR?

• DNA polymerase

• all 4 deoxyribonucleotides

• dsDNA template

• a pair of primers which bind to opposite polynucleotide strands and have their 3 ends pointing towards each other