TRANSCRIPTION - Poultry Science tracking of transcription in Eukaryotes: Transport of RNA to the cytoplasm from the nucleus of eukaryotic cells can be determined through pulse-chase

TRANSCRIPTION

1) No free 3'OH end needed for initiation.

2) No primer needed.

3) 5' 3' synthesis directionality.

4) DNA-dependent RNA polymerase copies from one single-stranded region of DNA (antisense strand).

5) Ribonucleoside triphosphate precursors.

6) DNA-RNA duplex less stable than DNA-DNA duplex. RNA falls away from transcription site.

5' 3'

3' 5'

5' 3'

DNA

mRNA

Coding or Sense StrandHas same sequence as mRNA transcript (except that is possesses T instead of U)

Template or Antisense StrandDirects synthesis of complementary mRNA transcript

transcription

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Triphosphate precursors

Transcription (ribonucleoside triphosphate precursors; rNTPs )

UTP UMP2P

CTP CMP2P

GTP GMP2P

ATP AMP2P

Differences in Gene Expression Between Prokaryotes and Eukaryotes

Prokaryotes Eukaryotes

1.Unwinding by gyrases 1. Possible unwinding byand helicases, but no gyrases and helicases, but nosingle strand binding proteins. single strand binding proteins.

2. All RNA species are 2. Three different RNAsynthesized by a single polymerases are responsibleRNA polymerase. for the different classes

of RNA molecules.

3. mRNA is translated 3. Translation is independentduring transcription from transcription.(coupled).

Prokaryotes Eukaryotes

4. First nucleotide is a 4. mRNA is processed beforepurine. transport to the cytoplasm,

where it is translated.Caps and tails are added,and internal portions oftranscript are removed(splicing).

5. Genes are contiguous 5. Genes are often split.segments of DNA that They are not contiguousare colinear with the segments of coding sequencesmRNA that is rather, the coding sequencestranslated into a are interrupted by interveningprotein. sequences (introns).

6. mRNAs are often 6. mRNAs are often monocistronic.polycistronic.

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3’ 5’termination

A. Prokaryotic – Ex. lac operon model.I = Repressor genelac = lactoseP = PromoterO = Operator

RNA polymerase (Holoenzyme) = core unit + σ (sigma unit) =

σ70 = vegetative, for all RNAs

σ60 = nitrogen metabolism

σ 32 = heat shock proteins

σ

Initiation:

a) Holoenzyme binds to promoter site, as specified by sigma subunit and unwinds short stretch of DNA.

b) Sigma subunit released and transcription begins past promoter site.

Termination:

a) rho-independent: rich G-C dyad region followed by series of A’s and U’s (6-8) on RNA transcript causes formation of hairpin structure and consequently causes RNA polymerase to be knocked off.

b) rho-dependent: rho protein (hexamer) binds with RNA transcript at rut site and causes it to fold around it, and upon transclocation and further wrapping pulls the transcript away from the polymerase.

5'

B. Eukaryotic-Different RNA polymerases, with no sigma units:RNA polymerase I - rRNARNA polymerase II - pre-mRNARNA polymerase III - tRNA and other small

structural RNAs

Eukaryotic RNA Polymerase

RNA polymerase I (inside nucleolus) - makes structural RNA (i.e., rRNA).

RNA polymerase II (in nucleoplasm) - makes heterogenous nuclear RNAs or hnRNAs (i.e., precursor mRNAs or pre-mRNAs).

RNA polymerase III (in nucleoplasm) - makes small adaptor RNAs (i.e., small nuclear RNAs and tRNAs).

Only one RNA polymerase in prokaryotes. However, itsactivity is much like that of RNA polymerase II in eukaryotes.

3' 5'TF TF

RNAPol II

Fig. 1

Initiation:interact(binding)

Trans-acting regulatory Cis-acting nucleotide sequencesproteins of two regulatory elements

1) Transcription Factors (TFs) 1) Promoters - bind TFs and RNAfunction in the positive Pol - Promoters are necessaryregulation of transcription but for transcription initiation.are not part of RNA polymerase 2) Enhancers - only bind TFs. TFs (RNA Pol). TFs contain bend or loop the DNA. This bringstwo functional domains: i) DNA - Distant Enhancers and Promotersbinding domain - binds selectively into close proximity to formto Promoters and Enhancers. activated transcription complexes -ii) trans- acting domain - involved increasing the overall transcriptional in protein - protein interactions rate. See Fig. 1.between TFs and between TFs andRNA Pol in the Promoter. See Fig. 1.

3' 5'TF TF

RNAPol II

Fig. 1

Promoter (30 – 120 bp long) – upstream of transcriptional start site

GC box

CAAT box

TATA box

-110bp

-70bp

Transcription start site

Pre-mRNA3' 5'

-30bp

+1bp

geneGGGCGG GGCCAATC TATAAA

In the Promoter region, the modular elements, TATA box, CAAT box, and GC box,bind TFs in order to stimulate transcription. See Fig. 2

Fig. 2Pre-mRNA being synthesized by RNA Pol II

Enhancers greatly increase the efficiency of transcription initiation. In contrast to Promoters, Enhancers have the following properties: i) Their positions and orientationsneed not be fixed, ii) They are at a greater distance from the target gene than arePromoters, and iii) They generically enhance the transcription of surrounding genes.

Termination:

1) rRNA (RNA Pol I) - Modified rho-independent. Strange G-C dyad region (many A's and U's intervening), followed by 26 A's, followed by 16 bases of any kind, followed by UCCACCUGGUC, followed by 3 bases, followed by AGGC.

2) pre-mRNA (RNA Pol II) - termination mechanism unknown. Processing begins before transcription ends. Possibly, processing proteins may literally pull pre-mRNA away and force RNA Pol II off of DNA.

3) tRNA (RNA Pol III) - standard rho-independent.

Three major aspectsof post-transcriptional processing

of primary transcripts of pre-mRNAs in eukaryotes.

RNA splicing - removal of non-coding intron (intervening) sequences, between coding exon (expressed) sequences.

Capping of 5' end (i.e., 7-methylquanosine) and addition of poly A tail to 3' end of pre-mRNA.

In eukaryotes, pre-mRNA must associate with proteins to get out of the nucleus and into the cytoplasm.

Four major aspects of post-transcriptionalprocessing of tRNAs in eukaryotes.

Cut at G on 5' end.Addition of CCA sequence at 3' end (where AA will attach).Base modifications (i.e., thymine, pseudouracil) in looped regions.Introns excised from terminal loop.

Aspects of post-transcriptionalprocessing of rRNAs in eukaryotes.

Introns removed by autocatalytic activity of EXON regions.213

Exon 1 Intron A Exon 2 Intron B Exon 3

Pulse-Chase tracking of transcription in Eukaryotes:

Transport of RNA to the cytoplasm from the nucleus of eukaryotic cells can be determined through pulse-chase studies using 3H-uridine. If a eukaryotic cell is exposed to radioactive RNA precursor for a few minutes, and the intracellular locationof the incorporated radioactivity is determined by autoradiography, almost all of the nascent, labeled RNA is found in the nucleus. If the short exposure “pulse” to the labeled RNA is followed by a period of growth in non-radioactive medium before autoradiography, almost all of the radioactivity is found in the cytoplasm.