L10Biol261W2013Transc Revised

8/9/2019 L10Biol261W2013Transc Revised

1/37

L10


2/37

A

A*

5

3

3 5

Replication of the region the top DNA strand labeledA would involve:a) Discontinuous synthesis of the lagging strandb) Discontinuous synthesis of the leading strand

c)

Continuous synthesis of the lagging strand

d) Continuous synthesis of the leading strand


3/37

Origin 5

..GATCCA.3

Direction of DNA polymerase III movement on this strandfragment (right or left) ? Complementary making and moving.What is the sequence of the leading template strand ?

Complementary base pairs

What is the sequence of the corresponding Okazaki fragment ?Complementary strand directions

Replisome structure andaccessory proteins arehighly conserved, fromvirus to eukaryotes


4/37

RNA is similar to DNA, but,1. RNA is usually single stranded,

2. The strand backbone is composed

of a ribose sugar, (2

OH),

not deoxyribose sugars

3. RNA contains the pyrimidine base

URACIL instead of thymine.

4.RNA can catalyze biological

reactions

3


5/37

RNA has a

sugar-phosphate backbone(phosphodiester bonds).

The ribose has a OH on

the 2

carbon.

RNA is single stranded.

RNA is has 5

to 3

polarity like DNA.

4


6/37

There are two grades and 6+ classes of RNA: pp 287

(1) Gene information-coding

genes (a)Messenger (mRNA)is the protein encoding transcript ofa gene(1% of RNA in humans)

(2) Functional RNA-may have catalytic propertiesgenes ? (b)Ribosomal RNA rRNA is part of the translation

complex.

genes ? (c) Transfer RNA tRNA participates in translation. Itcarries specific amino acids to be incorporated into the new protein.

genes? (d) Small nuclear RNAs(sn RNAs)- spliceosome, rRNA

assembly specific to eukaryotes.

genes? (e)Micro RNA

s - short 20-25 nucleotide bases, single

stranded RNA

s that may be involved in gene expression or mayblock the translation of mRNA.

(f)Small interfering RNAs (siRNA) exogenous double stranded RNA

19-25 bases

genes ? (g)Long noncoding RNA (lncRNA)- transcriptional control

(XIST) and epigenetic regulation (200 + bases).

5


7/37

RNA has (1) sugar-phosphate backbone

(2) 4 nucleotides (A U, C, G)

(3) directionalityIt is synthesized from the 5

end to the 3

end.

But, it is single stranded and has uracil instead of thyamine

Convention - DNA is always drawn with the upper strandrepresented in the 5 to 3 direction, mRNA same .

6


8/37

DNA is double stranded.

5-GCACTACGCATCGATCGACTAGCTAGCATC-33-CGTGATGCTTAGCTAGCTGATCGATGCTAG-5

The standard representation is the non template orcoding strand:

GCACTACGCATCGATCGACTAGCTAGCATC

Assume the sequencerepresents double stranded DNA

with the upper strand shown, in the 5 to 3 direction,

(unless text describes it otherwise).

7


9/37

DNA

RNA

mRNA is transcribed from DNA 5- 3 in the coding strand order

-The RNA producthas the same sequence as the upper, coding

strand of DNA,except in has Us in place of Ts,BUT

The lower strand of DNA is the physical template for RNA synthesis.

RNA is drawn in the 5

(left) to 3

(right) direction.

8


10/37

The 8thedition refers to the coding and template strands.

The 9th & 10thedition (white& black books ) refers to template and

non- template strands, which is called the coding strand(pp 289).

There is not always consistency between books or people

, be careful, check sequence and directionElsewhere you may see:

Template strand = sometimes coding strand, nonsense strand,

antisense strand

Non template strand =coding, non coding strand,sense strand

9VARIATION IN NOMENCLATURE


11/37

Gene function - TRANSCRIPTION

Gene transcription the primary transcript or pre

mRNA is synthesized or transcribed from the DNA

template 5

- 3

and may then be modified into the

message (mRNA)

transcription translation

DNA mRNA Protein (central dogma)

10

But we already know of 1 exception .


12/37

There are 3 stages of transcription:

Initiation, Elongation, Termination

11


13/37

12

A Promoteris a (small) region of (consensus) sequenceelements of DNA, which are necessary to initiatetranscription.

Conserved sequence-if several nucleotide sequences (amongspecies) align perfectly or close to it, - effectively the samenucleotide sequence.

Consensus sequence-if several sequences align but not soperfectly- there is some variation among sequences, but asignificant percentage of nucleotides co-occur at a highfrequency.

Initiation - Bacterial transcription is initiated (1) at apromoter sequence (2) when a RNA polymerase holoenzyme

binds to it.


14/37

Promoter sequences and the consensus sequence(promoters or

promoter elements)are on the 5

side (upstream) of of the

transcription start site (1+) coding strand

13


15/37

RNA polymerase is a multimericprotein complex .

E. coliRNA polymerase.

Prokaryote Initiation

-assembly of core enzyme, interactions with regulatory proteins

- catalysis -ribonucleoside triphosphate binding site- binds to DNA template, helicase activity

!- core enzyme assembly, regulation of gene expression

#- binding core enzyme to the promoter(position holoenzyme -10,-35),

strand separation,

Holoenzyme

(complete

enzyme)

core enzyme

14

One of

many

~10 bases


16/37

15

5

UTR(untranslated

region)

one of man


17/37

Eukaryotic Initiation


18/37

Eukaryotic DNA is more complicated than Prokaryotic

(1)Many more genes,

(2)

more DNA and

(3)there is more intervening (non message-coding) DNA in eukaryotes,

thus the gene density is lower in eukaryotes:1/1400 bp in E. coli

1 gene /9000 bp in Drosophila,

1 gene / 100,000 bp in humans

(4) chromatin structure plays a key role in gene transcription.

(5) more complicated cellular structure, different tissues etcTHUS , it is not surprising thatRNA polymerases are more

complicated in eukaryotes, requiring more polymerases, a more

complex promoter, accessory and regulatory proteins (transcription

factors)

16


19/37

In eukaryotic organisms:

-RNA polymerase Itranscribes rRNA

in the nucleolus, except for the small

5S rRNA (large fraction of

transcription).

-RNA pol II transcribes all protein

coding genes, some snRNA

s, in thenucleus,LncRNA.

-RNA pol IIItranscribes small

functional RNA genes such as those

in the spliceosome, 5S rRNA, transferRNA (tRNA),sn RNA

s not made by

RNA pol II in the nucleus

17


20/37

(1) Core promoterEukaryotic genes have a TATA (TATAAAA) box isabout -30 region and an initiator site which spans 1+, specifying

where the transcription polymerase assembles and beginsOther

promoter sites: -40 and -120 (GC), -80 (CAAT), -120 (Octamer)

(2) (a) There are many additional cis - regulatorysequences (activators100s bp + upstream only, enhancers ~ 1000 bp +, up and downstream)

(b) trans actingGeneral Transcription Factors (GTFs), and

(c) in vertebrates, particularly mammals, the absence of histone

methylation (nucleosomes)near the promoter allows expression.

Eukaryotic Initiation 19


21/37

Figure 4-57 Molecular Biology of the Cell( Garland Science 2008)

(1) Expression starts withunwinding DNA, starting withthe nucleosome, although it is inan extended form in G1, early G2

18

Extended form, local unwindingof nucleosomes


22/37

(1) Remove methyl tags and unwind nucleosomes

(2) TranscriptionBindingProteinat the TATA box - attracts other

GTFs (TBP is part of 1, of several GeneralTranscriptionFactors)+ RNA polymerase II core, forming thepre-initiation complex(3) Interaction of (upstream) cis-enhancer sequences

(4) Transcription Initiation

(5) Dissociation of GTP and Elongation

TBP

20Nucleosome wound, promoter methylated


23/37

Transcription initiation in eukaryotes 21


24/37

Transcription initiation in eukaryotes

TBP is part of TFII D protein (and several TBP associated factors)TFIID is one of several GTFs -general transcription factors

or Transcription Factor for RNA polymeraseII X(X=factor letter)

22


25/37


TBP attracts other GTF

s and then,

the RNApolymerase II coretogether the preinitiation complex

23


26/37


Transcription is initiated with the

phosphorlation of the Carboxyl

TailDomain,RNA polymerase

dissociates from most of the

GTF

s, but some remain at the

promoter-attracting the next core

enzyme

24


27/37

The bases in RNA are added in a sequence that is complementary

to the DNA sequence G opposite C

s

C opposite G

s

U opposite A

s

A opposite T

s

ELONGATION in General 25

Eukaryotes FACT (facilitates chromatin transcription)hetero dimer deals with histones


28/37

RNA polymerase opens the DNA duplex, RNA is synthesized in the

5

to 3

direction from one strand of DNA and then closes it. A single

gene is only transcribed in one direction.

Only one strand is the template for 1 geneChromosomes have different genes in different orientations, so

different strands may be transcribed for different genes at

different locations.26


29/37

27


30/37

There are transcription termination signals in the DNA, beyond theprotein coding sequence:

(1)Intrinsic- GC rich hairpin which disrupts DNA-RNA binding

(2)Rho(a helicase) binding site (rut=rho utilization site),rho

unwinds RNA&DNA facilitating RNA polymerase release.

ProkaryoteRNA transcriptiontermination28


31/37

Cotranscriptional processing of Eukaryotic RNA: capping

The initial RNA transcript is capped with a 7-methylguanosinetriphosphate.

Unusual the linkage is 5 to 5 and the three phosphates are

maintained, unlike RNA (or DNA) synthesis catalyzed by RNA

polymerase.

Carboxyl TailDomain

30


32/37

Cotranscriptional processing of Eukaryotic RNA (2)

2. Most eukaryotic genes have

blocks of coding(exons) and noncoding (intron) DNA

The mRNA is transcribed primary

transcript(pre mRNA) with the

introns and the exonsAnd the introns are spliced out

before the mRNA is translated.

31


33/37

Introns are looped out and are (1)cut at specific sequences

(exon-GU consensus sequence..intron..consensus sequenceAG-

exon), (2)removed and (3)the exons are spliced together to produce amature mRNA with a central coding region in red).

32


34/37

Transcriptional processing (3) Many human genes have

alternate splicing patterns several different related proteins

can be produced by one gene.

33products of several loci can also be spliced into one mRNA.


35/37

Cotranscriptional processing of RNA

Termination: when the highly

conserved sequenceAAUAAA or

AUUAAA is recognized , it signals a

termination enzyme to cut the end

~20 bases downstream and add a

polyA tail (AAA)- polyadenylation

signal

Capping

Intron removal

Splicing

34


36/37

The mRNA is cleaved about 20 bp after a polyadenylation signal

and a poly (A) tail of about 300 nucleotides is added to the 3

end

of the mRNA.

2. 35Coding RNA


37/37

Box 1. Key Genetic Features of Multicellular Organisms - S. B.

Caroll (2005) Evolution at 2 levels Plos Biology

Individual regulatory proteins function in many different contexts.

The expression of individual genes is multiply regulated, tissue-specific

and temporal controlled.

Many regulatory proteins are members of large families and can overlap

in function..

Multiple protein forms may be encoded by single genetic loci.Alternative

protein forms (isoforms) may function in different contexts and/or

possess different activities.

Documents

L10Biol261W2013Transc Revised