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Transcription

RNA polymerase molecules on DNA are

producing ribosomal RNA moleculesthat bind to ribosomal proteins.

Transcription starts at a "promoter."Transcription ends at a "terminator."

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A transcription unit = a gene or operon

Genes are tightly packed (some

overlapped) in bacterial genomes butloosely packed with many long intergenic

spaces in eukaryotic genomes.

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Bubble

Bubble

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Transcription cycleR Y R

+1

Promoter for E. coli 70-RPase

Holoenzyme (core + )

finds and binds a

promoter.

A region around -10

and +1 is unwound

to form a bubble.

interacts with the -35

and -10 hexamers.

Isomerization from

closed

to open binary complex

Nucleotides bind to form

ternary complex

Abortiveinitiation

cycling

Closecomplex

Open

complex

Elongation

complex

Termination

complex

Short RNAs are produced, as

the contact with -35 is lost.

Short RNAs are repetitively releasedand new RNAs are produced.

is released

and promoter

is cleared.

Elongation is

processive

and stable.

RNA is released

in termination

and RPase

dissociates

from DNA

Intrinsic terminators code for

RNA hairpin - oligo(U)

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Eukaryotic transcription

A RPase II promoter

Nuclear RPase I, II, III and IV (?),

Mitochondrial and chloroplast RPases

Initiation complex

TFIID binds and

bends TATA box.

Many initiation

factors are

loaded.

by TFIIH during

Abortive cycling

TFIID remains at

promoter and PRPasemoves down.

Many proteins (>100 subunits)

must assemble around the +1site.

The order of assembly varies

from gene to gene. Some

protein complexes are pre-

assembled before brought toDNA together.

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Eukaryotic mRNA processing in nucleus5'-UTR 3'-UTR

Exons + Introns

All the mRNA

processing events,

capping, splicing,

3'-end processing

occur during

transcription within

nucleus.

The processing

factors bind to CTDof RPase and act on

the mRNA parts

that are coming out

of the RPase.

Only RPase II hasCT

Capping at the 5' end of mRNA

5'-P-5'

linkage CBC (cap

bindingcomplex)

marks a

successfully

capped end.

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Eukaryotic mRNA splicingTwo sequential Exon = Expressed part, Intron = Intervening part

transesterification

reactions at a site

EJC (exon junction

complex) marks asuccessfully spliced site

5'-Splice

site

Branch

site

3'-Splice

site

The 5'-splice, branch and

3'-splice sites are highly

homologous in all splicings.

Splicing occurs sequentially

as soon as CTD-boundsplicing factors recognize

relevant sites sequentially

coming out of RPase II.Thus, a mature RNA of

exon3-exon1-exon2 cannot

be made in vivo.

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Spliceosome: snRNA-snRNA interactions

SRproteins

mark

exons.

RNA-RNA

Rearrange-

ments

using ATP

hydrolysis.

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Alternative and abnormal splicing Alternative splicing occursby skipping exons.

Diverse proteins are made from

alternatively spliced mRNAs,which all are made

from a gene.

Other forms of diversity

are alternative start sites

of transcriptionand translation.

Many of these are

subject to regulation.

Abnormal splicing can

occur by some mutationsor polymorphisms in DNA

that inactivate normal

splice sites or activate

cryptic splice sites.

Some are associated withdiseases.

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Self-splicingTwo sequential

transesterification

reactions at a site

Self-splicing RNA

has to fold into a

proper structurewithout the help of

spliceosome.

Thus, most of the

intron sequences

are critical in

selfsplicing,

whereas

they are not critical

in spliceosome

splicing.

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Processing at 3' end and nuclear transport

3'-Cleavage and

polyadenylationPoly(A) polymerase does not

require a template.

After 3'-cleavage, RPase II

continues for some distance.

Poly(A)-binding proteins mark polyadenylation.Nuclear export receptor is loaded on mature mRNA.

RNA debris are degraded by nuclear exosome.

Nuclear transport

"export-ready nuclear pore

RNA complex

CPSFCstF

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Non-coding RNA synthesis and processing in nucleolus

In nucleolus, some ribonucleoprotein

complexes (RNAs + proteins) areassembled (e.g. ribosomal subunits,

U6 snRNP, telomerase, signal

recognition particle, etc.) and tRNAs

are processed.

snoRNA (encoded by introns) and snRNA

produced mostly by RPase II aremodified at Cajal bodies and GEMS.

In nucleolus, pre-rRNA is produced

by RPase I, modified, cleaved into 3

rRNAs, and complexed withribosomal proteins.

(5S rRNA is produced by RPase III

and not modified.)

Subnuclear structures

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Figure 6-47. The function

of the nucleolus in

ribosome and other

ribonucleoproteinsynthesis.

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Terms:CTD carboxyl terminal domain

SR proteins -serine/arginine rich proteins

hnRNPs -heterogeneous nuclear ribonuclear proteinsCstF -cleavage stimulation factor F

CPSF -cleavage and polyadenylation specificity factor

Additional subnuclear structures:1.Cajal bodies (named for scientist who first described them in 1906)

2.GEMS (Gemini of coiled bodies)

-resemble one another and are frequently paired in the nucleu

-may be sites where snRNAs and snoRNAs undergo their final

modifications and assembly with protein-also sites where the snRNPs are recycled and their RNAs

are reset after the rearrangements that occur during splicing

3. interchromatin granule clusters (also called speckles)

-stockpiles of fully mature snRNPs that are ready to be used ins licin of re mRNAs