BIOL 2301 Ch. 13

central dogma

DNA → RNA through transcription

reverse transcription exists

occurs inside the nucleus

mRNA leaves nucleus, finds ribosome and is read, product is assembling a chain of amino acids (polypeptide), folds into active protein

an early RNA world

living organisms must be able to store and faithfully transmit genetic info

must have the ability to catalyze chem transformation that drive life processes

used to think these were handled by nucleic acids and protein enzymes

Cech discovered that RNA can serve as a biological catalyst

DNA eventually replaced RNA as the primary carrier of info

• more stable

RNA

ribozymes

evidence suggests

RNA was og genetic material

ribozmes

catalytic RNA

structure of RNA

primary

secondary

single polynucleotide strand

primary RNA structure

linear sequence of ribonucleotides

only ribose sugars

only uracil, no thymine

tries to acquire a secondary structure to protect bases

secondary RNA structure

forced to protect bases

hydrogen bonds formed with itself

useful for termination of transcription sometimes

classes of RNA

rRNA

mRNA

tRNA

snRNA’s

snoRNA’s

scRNA

miRNA

siRNA
piRNA

lncRNA

crRNA

rRNA

doesn’t contain info to make proteins

makes machines to make proteins

2-4 pieces of this are assembled based on species

mRNA

contains info of what amino acids have to be constructed to make a protein

pre mRNA

• immediate products of transcription

• larger precursor molecules

• modified extensively before becoming mRNA

tRNA

super stable

tons of loops and turns

50% of bases are double stranded bc of secondary structure

reads what mRNA says and tells what amino acid goes where

attaches to 1 type of amino acid and helps incorporate it into a polypeptide chain

snRNA

small nuclear

combine with small protein subunits to from snRNP’s

snoRNA

small nucleolar

take part in processing of rRNA

scRNA

small cytoplasmic

miRNA

microscopic

carry out RNA interference

siRNA

small interfering

piRNA

piwi interacting

similar to miRNA and siRNA

role in suppressing expression of transposable elements

lncRNA

long non-coding

variety of functions

regulation of gene expression

crRNA

CRISPR

assist in destruction of foreign DNA molecules

transcription requires

3 major elements

DNA template

raw materials (ribonucleotide triphosphates)

transcription apparatus

DNA template

transcribed stand = template

transcription unit

• promoter

• RNA coding sequence

tree trunk structure

3’-5’ to make 5’-3’

no okazaki

double stranded

promoter of transcription unit

tells machinery to bind and start transcribing

serves as sequences to which transcription apparatus binds

determines 1st nucleotide that’s transcribed into RNA

determines which DNA strand is the template

initiation

initiation in promoter

substrate for transcription

• ribonucleoside triphosphates - rNTP’s added to 3’ of RNA, substrates used in RNA synthesis

• transcription apparatus

RNA coding sequence of transcription unit

sequence of DNA that copies into an RNA molecule

tree trunk structure of DNA template

thin central fiber that has attached strings that have granules

thin central fiber = DNA molecules

strings = RNA

transcription begins at top, where RNA is short

terminator

terminator

tells transcription to stop

usually a part of the RNA coding sequences

transcription apparatus

consists of proteins necessary for catalyzing the synthesis of RNA

diff in bacteria and eukaryotes

nucleotides always added to 3’ end of RNA

bacterial transcription apparatus

bacterial RNA polymerase

large multimeric enzyme

5 subunits

• 2 alphas

• beta

• beta’

• omega

core catalyzes the elongation of RNA

sigma factor controls binding of RNA polymerase to promoter

• associates with core to make a holoenzyme

eukaryotic transcription apparatus

3 types

• RNA polymerase 1

• RNA polymerase 2

• RNA polymerase 3

found in all eukaryotes

in plants, also a 4 and 5

RNA polymerase 1

eukaryotic

transcribes RNA

RNA polymerase 2

eukaryotic

transcribes pre-mRNA’s, snoRNA’s, miRNA’s, and smRNA’s

RNA polymerase 3

eukaryotic

transcribes other small RNA’s

from DNA to protein

sequence of RNA is reverse compliment of template

template connected to coding strand

transcription starts right after promoter

all cellular RNA transcribed from DNA

substrate for transcription

RNA synthesized from rNTP’s

• has ribose sugar and a base attached to 3 phosphate groups

nucleotides added one at a time to 3’ OH of growing nucleotide

2 phosphate groups cleaved from incoming strand

remaining phosphate group participated in a phosphodiester bond

RNAn + rNTP → RNAn + 1 + Ppi

3 stages of bacterial transcription

initiation

elongation

termination

initiation in bacterial transcription

transcription apparatus assembles on promoter and begins synthesis of RNA

includes

• promoter recognition

• formation of transcription bubble

• created of first bond between rNTP’s

• escape of transcription apparatus from promoter

bacterial promoters

initial RNA synthesis

bacterial promoters

essential info for transcription apparatus embedded

usually adjacent to RNA coding region

consensus sequence

consensus sequence

set of most commonly encountered nucleotides among sequences that possess considerable similarity

most common one is -10

another common one is -35

pribnow box

being A-T rich helps minimize energy needed to melt bubble

base substitutions in these reduce transcription

• down mutations

• slow down

holoenzyme binds to these

upstream element

• contains A-T pairs at around -40 - -60

-10 consensus sequece

TATAAT

-35 consensus seqeunce

TTGACA

pribnow box

5’ TATAAT 3’

3’ ATATTA 5’

initial RNA synthesis

once holoenzyme bound to promoter, RNA polymerase positioned over transcription start site

position of start site determind by locations of consensus sequences

RNA polymerase pairs at base of start site on template

no primer needed

abortive initiation

• RNA polymerases repeatedly generate and release short transcripts while still bound to promoter

• occurs in both pro and euks

elongation in bacterial transcription

RNA polymerases undergoes change in conformation and no longer able to bind to consensus sequences

• allows polymerase to escape promoter and begin transcribing downstream

as polymerase moves downstream, progressively unwinds DNA at leading (downstream) edge of bubble

transcription bubble

transcriptional pausing

accuracy of transcription

• RNA polymerase capable of proofreading in the course of transcription

transcription bubble

transcription takes place within a short stretch of unwound DNA

RNA continuously synthesized here

as transcription apparatus moves down template, generates positive supercoiling ahead and negative behind

transcriptional pausing

caused by backtracking

• RNA polymerase slides backwards

• disengages 3’ OH

• temporarily halts further RNA synthesis

• several mechanisms to prevent

  • proteins that cleave backtracked RNA

• important in transcriptional proofreading

important in coordination of transcription and translation in bacteria

also affects rate of RNA synthesis

termination in bacterial transcription

most terminators found upstream of site where it actually happens

like a car that stops after bump

several events needed

2 major types

polycistronic mRNA

2 major types of termination

rho-dependent

rho-independant

rho-dependent termination

able to cause termination only in presence of ancillary protein called rho-factor

2 features

• DNA sequences of terminator

• DNA sequence upstream of terminator

when polymerase encounters terminator, pauses and allows rho to catch up

rho factor has helicase activity

• unwinds DNA-RNA hybrid

DNA sequences of terminator

causes RNA polymerase to pause

DNA sequence upstream of terminator

encodes a stretch of RNA that’s usually rich in C and devoid of secondary structures

rho-utilization site serves as binding site for rho-factor

once rho ninds to RNA, moves towards 3’

rho-independent termination

able to cause termination in absence of rho-factor

multistep process

50% of all terminators in prokaryotes

2 features

• inverted repeats

• a string of 7-9 A nucleotides

inverted repeats

rho-independent

sequences of nucleotides on same strand that are inverted and complementary

when transcribed into RNA, form a hairpin

string of 7-9 A nculeotides

rho-independent

follows inverted repeats

transcription of this produced a string of U after hairpin in transcribed RNA

• causes polymerase to pause, allowing hairpin to form

  • destabilizes DNA-RNA pairing

polycistronic mRNA

a group of genes often transcribed into a single RNA
produced when a single terminator is present at the end of a group of several genes that are transcribed tg

uncommon in eukaryotes

eukaryotic transcription similar to bacterial

includes initiation, elongation, and termination

also has basic principles of transcription already outlined

eukaryotic cell possess 3 diff RNA polymerases

each transcribes a diff class of RNA and recognizes a diff promoter

generic promoter can’t be described for eukaryotic cells

depends on whether it’s recognized by polymerases 1-3

transcription and nucleosome structure

requires that sequences on DNA be accessible to RNA polymerase and other proteins

chromatin structure modified before transcription so DNA is in a more open configuration and more accessible

chromatin remodeling proteins can bind to chromatin and displace nucleosomes from promoters

promoters

holoenzyme recognizes and binds directly to sequences in promoter

transcription factors

promoter for gene transcribed by polymerase 2, has 2 primary parts

polymerase 1 and 3 promoters

transcription factors in promoters

bind to DNA and affect levels of transcription

general transcription factors

• combines with polymerases to form

basal transcription apparatus

• group of proteins that assembles near transcription start site

• sufficient to initiate minimal levels of transcription

2 primary parts of promoter

core

regulatory

core of promoter

located immediately upstream of gene

larger than regulatory

site where basal transcription apparatus binds

TF2B recognition element (BRE)

one or more consensus sequences

• TATA box most common

  • -25 to -30 upstream of start site

initiator element

downstream core promoter element

• recognized by certain transcription factors

  • bind to them and serve as a platform for assembly of basal transcription apparatus

regulatory part of promoter

immediately upstream of core

variety of consensus sequences

• transcription factors bind to these and make contact with basal transcription apparatus

  • affect rate at which transcription is initiated

transcription factors can also bind to distant sequences to enhancers

DNA between an enhancer and promoter loops out so transcription factors bound to enhancer can interact with basal

can activate and inhibit transcription

transcriptional sensor

polymerase 1 and 3 pormoters

distinct from polymerase 2

some in 3 contain internal promoters

• downstream of start site and transcribed into RNA

initiation of eukaryotic transcription

through assembly of transcription machinery on promoter

machinery consists of polymerase 2 and a series of transcription factors that form a giant complex of 50 or more polypeptides

machinery assembly begins when regulatory proteins bind DNA near the promoters and modify the chromatin structure so transcription can take place

basal consists of polymerase 2

1st step is binding TF2D to TATA box on core

after polymerase and general transcription factors are on core promoter

template strans positioned within active site of polymerase, creates an open complex

elongation of eukaryotic transcription

Kornberg got Nobel prize for discovering how eukaryotic RNA polymerase 2 functions during this

polymerase maintains transcription bubble during this

• 8 nucleotides of growing molecule remain paired with template strand

• double helix eneters cleft in polymerase and is gripped by extensions of enzyme

• DNA in unwound, complementary nucleotides are added

• as it funnels through polymerase, DNA-RNA hybrid hits a wall of amino acids and bends at almost a right angle

  • positions end of DNA-RNA hybrid at active site of polymerase where new nucleotides are added

termination in eukaryotic transcription

polymerase use different mechanisms for termination

1 requires a termination factor similar to rho factor used in terminating the transcription of some bacterial genes

binds to downstream DNA sequence

3 ends transcription after transcribing a terminator sequence that produces a string of U in RNA

• secondary structures often occur upstream of string of U and are necessary for transcription

• uses a termination mechanism similar to rho-indep termination in bacteria

2 does not occur at a specific sequences

• often continues to synthesize RNA hundreds-thousands of nucleotides past the coding sequence necessary to produce the mRNA

• pre-mRNA cleaved at a specific site designated by consensus sequence while transcription is taking place

  • cuts it into 2 pieces, mRNA that will encode the protein and the piece that at 5’ end trailing out of the polymerase

    • an enzyme called rat1 attaches to 5’ ends and moves toward 3’ end

RNA interference

eukaryotic gene expression can be downregulated by selectively targeting specific cytoplasmic mRNA’s

high abundance, short, non-coding RNA in the cytolasm are required for these regulatory mechanisms

2 types

• miRNA

• siRNA

regulatory RNA’s work in concert with other cytoplasmic factors in 2 ways

• inducing cleavage of cytoplasmic mRNA, leading to its degradation

• inhibiting translation initiation

regulation collectively known as RNAi