Transcription Transcription

transcriptiontranscription

Gene sequence (DNA) recopied or Gene sequence (DNA) recopied or transcribed to RNA sequencetranscribed to RNA sequence

Products of TranscriptionProducts of Transcription

1. Ribosomal RNA (rRNA)1. Ribosomal RNA (rRNA) - - Several Several rRNAs arerRNAs are

vital constituents of vital constituents of ribosomesribosomes

22 Transfer RNA (tRNA)Transfer RNA (tRNA) - - The molecule thatThe molecule that

physically couples nucleic acid codons with physically couples nucleic acid codons with specific amino acids specific amino acids

33 Messenger RNA (mRNA)Messenger RNA (mRNA) - - The nucleic The nucleic acidacid

messenger that carries encoded information messenger that carries encoded information from genes on DNA to the protein from genes on DNA to the protein manufacturing ribosomesmanufacturing ribosomes

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Transcription requires:Transcription requires: ribonucleoside 5´ triphosphates: ribonucleoside 5´ triphosphates:

ATP, GTP, CTP and UTP ATP, GTP, CTP and UTP bases are adenine, guanine, cytosine and bases are adenine, guanine, cytosine and

uraciluracil sugar is ribose (not deoxyribose)sugar is ribose (not deoxyribose)

DNA-dependent RNA polymeraseDNA-dependent RNA polymerase Template (sense) DNA strandTemplate (sense) DNA strand Animation of transcriptionAnimation of transcription

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Features of transcription:Features of transcription: RNA polymerase catalyzes sugar- catalyzes sugar-

phosphate bond between 3´-OH of ribose phosphate bond between 3´-OH of ribose and the 5´-POand the 5´-PO44. .

Order of bases in DNA template strand Order of bases in DNA template strand determines order of bases in transcript.determines order of bases in transcript.

Nucleotides are added to the 3´-OH of Nucleotides are added to the 3´-OH of the growing chain.the growing chain.

RNA synthesis does not require a primer.RNA synthesis does not require a primer.

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In prokaryotes transcription and In prokaryotes transcription and translation are coupled. Proteins are translation are coupled. Proteins are synthesized directly from the synthesized directly from the primary transcript as it is made.primary transcript as it is made.

In eukaryotes transcription and In eukaryotes transcription and translation are separated. translation are separated. Transcription occurs in the nucleus, Transcription occurs in the nucleus, and translation occurs in the and translation occurs in the cytoplasm on ribosomes.cytoplasm on ribosomes.

RNA PolymeraseRNA Polymerase DNA-dependent DNA-dependent

DNA template, ribonucleoside 5´ triphosphates, DNA template, ribonucleoside 5´ triphosphates, and Mgand Mg2+2+

Synthesizes RNA in 5´ to 3´ directionSynthesizes RNA in 5´ to 3´ direction E. coliE. coli RNA polymerase consists of 5 subunits RNA polymerase consists of 5 subunits Sigma factors are a subunit of RNA Sigma factors are a subunit of RNA

polymerase.polymerase. Sigma factors are needed for promoter Sigma factors are needed for promoter

binding, but after transcription starts they binding, but after transcription starts they

dissociate.dissociate.

Eukaryotes have three RNA Eukaryotes have three RNA polymerasespolymerases RNA polymerase II is responsible for RNA polymerase II is responsible for

transcription of protein-coding genes transcription of protein-coding genes and some snRNA moleculesand some snRNA molecules

RNA polymerase II has 12 subunits RNA polymerase II has 12 subunits Requires accessory proteins Requires accessory proteins

(transcription factors)(transcription factors) Does not require a primerDoes not require a primer

Stages of TranscriptionStages of Transcription

Promoter Recognition Chain Initiation Chain Elongation Chain Termination

promoter recognitionpromoter recognition Transcription factors bind to promoter s

equences and recruit RNA polymerase..

DNA is bound first in a DNA is bound first in a closed complexclosed complex. . Then, RNA polymerase denatures a 12–Then, RNA polymerase denatures a 12–15 bp segment of the DNA (open 15 bp segment of the DNA (open complex).complex).

The site where the first base is The site where the first base is incorporated into the transcription is incorporated into the transcription is numbered “+1” and is called the numbered “+1” and is called the transcription start sitetranscription start site..

Defined regions are transcribed

upstream region

transcribedregion

downstream region

promoter(RNA polymerase

binding site)

transcriptionstart site

terminationsite

gene dsDNA

TB

Transcription factors that are Transcription factors that are required at every promoter site for required at every promoter site for RNA polymerase interaction are RNA polymerase interaction are called basal transcription factors.called basal transcription factors.

promoter sequencespromoter sequences Promoter sequences vary considerably.Promoter sequences vary considerably. RNA polymerase binds to different RNA polymerase binds to different

promoters with different strengths; binding promoters with different strengths; binding strength relates to the level of gene strength relates to the level of gene expressionexpression

There are some common There are some common consensus sequences for promoters: for promoters: Example: Example: E. coliE. coli –35 sequence (found 35 bases 5 –35 sequence (found 35 bases 5

´ to the start of transcription)´ to the start of transcription) Example: Example: E. coliE. coli TATA box (found 10 bases 5´ to TATA box (found 10 bases 5´ to

the start of transcription)the start of transcription)

“-35 squence” “Pribnow box (-10 Sequence)

Ptrp TTGACA----17bp----TTAACTA---transcriptionPlac uv5 TTTACA----18bp----TATAATG---transcriptionPtac TTGACA----16bp---TATAATG---transcriptionProkaryotic TTGACA TATAATconsensusHuman ß-globin CCAAT-----39bp----CATAAA----transcriptionEukaryotic CCAAT ATAconsensus

Sekuen DNA dan beberapa promotor bakteri

enhancersenhancers

Eukaryotic genes may also have Eukaryotic genes may also have enhancers.enhancers.

Enhancers can be Enhancers can be located at great at great distances from the gene they regulate, distances from the gene they regulate, either 5´ or 3´ of the transcription start, either 5´ or 3´ of the transcription start, in introns or even on the noncoding in introns or even on the noncoding strand.strand.

One of the most common ways to One of the most common ways to identify promoters and enhancers is to identify promoters and enhancers is to use a reporter gene.use a reporter gene.

other playersother players Many proteins can regulate gene Many proteins can regulate gene

expression by modulating the strength of expression by modulating the strength of interaction between the promoter and interaction between the promoter and RNA polymerase.RNA polymerase.

Some proteins can activate transcription Some proteins can activate transcription (upregulate gene expression).(upregulate gene expression).

Some proteins can inhibit transcription by Some proteins can inhibit transcription by blocking polymerase activity.blocking polymerase activity.

Some proteins can act both as repressors Some proteins can act both as repressors and activators of transcription.and activators of transcription.

chain initiationchain initiation RNA polymerase locally denatures the RNA polymerase locally denatures the

DNA.DNA. The first base of the new RNA strand is The first base of the new RNA strand is

placed complementary to the +1 site.placed complementary to the +1 site. RNA polymerase does not require a RNA polymerase does not require a

primer.primer. The first 8 or 9 bases of the transcript are The first 8 or 9 bases of the transcript are

linked. Transcription factors are released, linked. Transcription factors are released, and the polymerase leaves the promoter and the polymerase leaves the promoter region.region.

RNA Pol.

InitiationInitiation

T. F.

RNA Pol.

5’RNA

Promoter

T. F.

T. F.

chain elongationchain elongation

RNA polymerase moves along the RNA polymerase moves along the transcribed or template DNA strand.transcribed or template DNA strand.

The new RNA molecule (primary The new RNA molecule (primary transcript) forms a short RNA-DNA transcript) forms a short RNA-DNA hybrid molecule with the DNA hybrid molecule with the DNA template.template.

chain terminationchain termination

Most known about Most known about bacterial chain termination

Termination is signaled by a Termination is signaled by a sequence that can form a hairpin sequence that can form a hairpin loop.loop.

The polymerase and the new RNA The polymerase and the new RNA molecule are released upon molecule are released upon formation of the loop.formation of the loop.

UUUURNA

3' end of RNA

Termination siteTermination site

Rho and TerminationRho and Termination

Rho independentRho independent termination depends termination depends on both slowing down the elongation on both slowing down the elongation complex, and an AT rich region that complex, and an AT rich region that destabilizes the elongation complexdestabilizes the elongation complex

Rho dependentRho dependent requires a protein requires a protein called called RhoRho, that binds to and slides along , that binds to and slides along the RNA transcript. The terminator the RNA transcript. The terminator sequence slows down the elongation sequence slows down the elongation complex, complex, Rho Rho catches up and knocks it off catches up and knocks it off the DNAthe DNA

RNAPol.

5’RNA

RNA Pol.

5’RNA

RNA Pol.

5’RNA

TerminationTerminationRho IndependentRho Independent

Terminator

RNAPol.

5’RNA

TerminationTerminationRho DependentRho Dependent

Terminator

RNA Pol.

5’RNA

RNA Pol.

5’RNA

Help, rhohit me!

mRNA mRNA synthesis/processingsynthesis/processing

Prokaryotes: mRNA transcribed directly Prokaryotes: mRNA transcribed directly from DNA template and used from DNA template and used immediately in protein synthesisimmediately in protein synthesis

Eukaryotes: primary transcript must be Eukaryotes: primary transcript must be processed to produce the mRNA to produce the mRNA Noncoding sequences (introns) are removedNoncoding sequences (introns) are removed Coding sequences (exons) spliced togetherCoding sequences (exons) spliced together 5´-methylguanosine cap added5´-methylguanosine cap added 3´-polyadenosine tail added3´-polyadenosine tail added

mRNA mRNA synthesis/processingsynthesis/processing

Removal of introns and splicing of exons can Removal of introns and splicing of exons can occur several waysoccur several ways For introns within a nuclear transcript, a For introns within a nuclear transcript, a

spliceosome is required. spliceosome is required. Splicesomes protein and small nuclear RNA (snRNA)Splicesomes protein and small nuclear RNA (snRNA) Specificity of splicing comes from the snRNA, some of Specificity of splicing comes from the snRNA, some of

which contain sequences complementary to the splice which contain sequences complementary to the splice junctions between introns and exonsjunctions between introns and exons

Alternative splicing can produce different forms of a Alternative splicing can produce different forms of a protein from the same geneprotein from the same gene

Mutations at the splice sites can cause diseaseMutations at the splice sites can cause disease ThalassemiaThalassemia • Breast cancer (BRCA 1)• Breast cancer (BRCA 1)