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transcription Cheat Sheet (DRAFT) by

jekslbfowjbgksjbg kejthketn

This is a draft cheat sheet. It is a work in progress and is not finished yet.

nucleotide & nucleic acids

nucleotide
sugar + N base + phosphate backbone
nucleoside
^ - phosphate
nucleotide functions
energy for metabolism (ATP)
 
enzyme cofactors (NAD+)
 
signal transd­uction (cAMP)
nucleic acid functions
storage of DNA
 
transm­ission of DNA
 
processing of ribozymes
 
protein synthesis
 
regulation of expression
DNA & RNA are polymers of nucleotide subunits which are linked by phosph­odi­ester bonds

DNA structure

two chains of nucleo­tides coiled around each other in a right-­handed double helix
sugar-­pho­sphate backbones of two strands spiral around outside of helix
N bases extend into centre at right angles to the acids of helix
adenine forms 2 H bonds with thymine
cytosine forms 3 H bonds with guanine
opposite polarity of two strands

RNA types

rRNA
80%
120 -5070 nucleo­tides
tRNA
15%
75 nucleo­tides
mRNA
varies
varies

RNA molecules

mRNA
interm­ediates that carry genetic inform­ation from DNA to ribosomes
tRNA
adaptors between amino acids & codons in mRNA
rRNA
structural & catalytic components of ribosomes
siRNA
RNA interf­erence
long non-coding RNA
transc­ription
microRNAs
RNA interf­erence
ribozymes
RNA enzymes
small nuclear RNAs
structural components of splice­osomes
tRNA -> ribozymes are non protei­n-c­oding
siRNA -> ribozymes are regulatory

RNA structure

intrin­sically single stranded
unable to form B-form helix due to bulky 2'-OH : when helical, RNA adopts A-form geometry, deep & narrow major groove, wide minor groove
secondary structure observed in rRNA & tRNA, & assumed to be in mRNA
mRNA carries instru­ctions for building a protein, eukaryotic mRNA is capped, polyA tail is not coding & is added after transc­ription to stabilise mRNA - its removal degrades RNA & inhibits transl­ation

rRNA

makes up ribosomes
ribosomes are protein factories in large macrom­ole­cular assemb­lies, composed of many proteins rRNA molecules
nucleolus is site of rRNA synthesis & ribosome assembly
ribosomal components are commonly designated by their 'S' values = rate of sedime­ntation in an ultace­ntr­ifuge
although 18s & 28s rRNAs of the eukaryotic ribosome contain many extra nucleo­tides not present in their bacterial counte­rparts, these nucleo­tides are present as multiple insertions that form extra domains & leave basic structure of each rRNA largely unchanged
transfer RNA is an interm­ediary between nucleic acid & protein worlds, acts as a translator

transc­ription in eukaryotes

RNA polymerase I
Synthe­sises pre-ri­bosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs)
RNA polymerase II
Respon­sible for synthesis of mRNA
RNA polymerase III
Makes tRNAs and some small RNA products
assembly of RNA polymerase
initiated by intera­ction of TATA-b­inding protein (TBP) with the promoter, two TF’s bind (IIA & IIB) ,TFIIE and TFIIH bind: TFIIF binds to RNA Pol and targets it to the promoter, TFIIE thought to be involved in DNA melting, Helicase activity in TFIIH unwinds DNA at the promoter
RNA strand initiation & promoter clearance
Kinase activity in TFIIH phosph­ory­lates the polymerase allowing the latter to escape the promoter, Initially 60-70 RNA nucleo­tides are synthe­sised, Then TFIIE & TFIIH are released
elonga­tion, termin­ation & release
TFIIF remains attached to RNA Pol II, Elongation factors help effici­ency, EF stop pausing and regulate post-t­ran­scr­ipt­ional proces­sing, phosphate removed at termin­ation

RNA processing

Almost all newly synthe­sised RNA molecules (primary transc­ripts) are processed to some degree in eukaryotic cells
The 5’-end is capped with methyl­gua­nosine
Introns are spliced out
Poly-A tail is built at the 3’ end - it probably protects 3’ end from enzymatic destru­ction. However some bacteria acquire Poly A tails but these promote destru­ction.

capping of 5' of mRNA

protects mRNA from 5’exon­uclease degrad­ation
Cap is 7-meth­ylg­uan­osine linked to 5’ end of mRNA
Formed by conden­sation of GTP with 5’ end of mRNA
Guanine is then methylated
Occurs early in transc­ription
capping enzymes are tethered to the c-terminal domain of polymerase II

placing poly (A) tail on mRNA

Pol II synthe­sises RNA up to and beyond the highly conserved seq: (5′)AAUAAA
An endonu­clease cleavage signal seq is bound by an enzyme complex
The RNA is cleaved by the endonu­clease at a point 10-30 nucleo­tides 3′ to (downs­tream of) the sequence AAUAAA
The polyad­enylate polymerase synthe­sises a poly(A) tail

transc­ription & RNA processing

the central dogma of biology is that inform­ation stored in DNA is transf­erred to RNA molecules during transc­ription & to proteins during transl­ation. inform­ation stored in the nucleotide sequences of genes is translated into the aa seqs of proteins through unstable interm­edi­aries (mRNAs). the mRNA codons on mRNA are translated into an aa seq by the ribosomes
in eukary­otes, the primary transcript is pre-mRNA. it is modified at both ends & introns are removed to produce mRNA. it is then exported to cytoplasm for transl­ation by ribosomes.
in RNA synthesis, the precursors are ribonu­cle­oside tripho­sph­ates, only 1 strand of DNA is used as template & RNA chains can be initiated de novo (without primer). RNA molecule will be comple­mentary to DNA antisense (template) strand & identical to DNA sense (non-t­emp­late) strand). catalysed by RNA polyme­rases & proceeds in 5' to 3' direction

transc­ription vs DNA replic­ation

RNA does not remain H-bonded to DNA post-s­ynt­hesis
RNA molecules are selective copies of shorter DNA segments
both employ polyme­rases - to make phosph­odi­ester linkages
DNA is unwound ahead of synthesis
similar building blocks
RNA polymerase does not need a primer
DNA in a human chromosome can be up to 250 million bases whilst most RNA molecules are a few thousand bases in length.
RNA polymerase makes an error 1 x 104 nucleo­tides compared to 1 x10 7 for DNA polyme­rase. As RNA is temporary it is not so critical. Still RNA polymerase has a proof reading mechanism.
the 'trans­cri­ption bubble' - because of unwinding & rewinding there are positive supercoils ahead of the bubble & negative behind. topois­ome­rases deal with positive supercoils & regulate negative ones.

transc­ription in prokar­yotes

stages
1. RNA chain initia­tion, 2. RNA chain elonga­tion, 3. RNA chain termin­ation
e.coli RNA polymerase
core enzyme = alpha2­-be­ta-­bet­a'-­omega
 
holoenzyme = alpha2­-be­ta-­bet­a'-­ome­ga-­sigma
 
alpha = assembly of the tetrameric core
 
beta = ribonu­cle­oside tripho­sphate binding site
 
beta' = DNA template binding region
 
sigma = initiation of transc­ription
promoters
must be >12 bp in e.coli to avoid occurrence by chance, have only small conser­vation in sequence
 
Startp­oint, -10 sequence (Pribnow box), -35 sequence and, the 17 nucl spacer seq between -10 & -35 seqs
 
70 bases in length before start point & 30 after.
transc­ription unit numbering
initiation site is +1
 
Bases preceding the initiation site are given minus (–) prefixes and are referred to as upstream sequences
 
Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences
1. binding & initiation
Binding of RNA polymerase holoenzyme to a promoter region in DNA
 
Localised unwinding of both DNA strands (around -10 region) by RNA polymerase to provide a single­-st­randed template
 
Formation of phosph­odi­ester bonds between the first few ribonu­cle­otides in the nascent RNA chain
 
Confor­mat­ional change in enzyme, promoter is cleared - sigma factor released
 
Nus A protein binds instead, ready for elongation - 'antit­erm­ination complex'
sigma cycle
RNA polyme­rase, guided by a bound sigma subunit, binds to DNA at a promoter sequence. Once RNA synthesis is initiated, the sigma subunit dissoc­iates stocha­sti­cally and is replaced by NusA. When RNA polymerase reaches a terminator sequence, RNA synthesis halts, NusA dissoc­iates from the polyme­rase, and the RNA polymerase dissoc­iates from the DNA. The free polymerase can, in principle, bind any sigma subunit. The type bound determines the promoter to which the RNA polymerase will bind in the next round of synthesis.
2. elongation
RNA polymerase is bound to DNA & is covalently extending the RNA chain, moves downst­ream.
 
Transc­ription bubble is about 18 nucleo­tides pairs and about 40 bases are added per second. Only about 3 bases are base paired at any moment in time.
3. termin­ation
RNA polymerase transc­ribes until it meets a termin­ator, Transc­ription then stops & the RNA product disass­ociates from the DNA template, Many termin­ators are hairpin forming sequences
 
Rho-de­pendent termin­ators — require a protein factor
 
Rho-in­dep­endent termin­ators — do not require protein factor
rho-in­dep­endent termin­ation
G-C rich stem, 7-9 bases after loop is U-run, U-DNA pairing is very weak allows dissoc­iation
rho-de­pendent termin­ation
(rho factor) 46-kD protein, active as a hexamer, Seqs for the few Rho termin­ators are 50-90bp, Rho binds to RNA
 
(hot pursuit model) - It binds to RNA tail and moves along transcript until it catches the polyme­rase, Rho has helicase activity causing RNA-DNA to separate

introns

- non-coding seqs located between coding sequences
- removed from the pre-mRNA and are not present in the mRNA
- Exons (both coding and non-coding sequences) are composed of the seqs that remain in the mature mRNA after splicing
- Introns are variable in size and may be very large