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Genetic Information Flow: Transcription of Class I, II, III Genes . YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE THE DEPARTMENT OF MEDICAL BIOLOGY ASST. PROF. DR. ENDER ŞİMŞEK. Transcription. Initiation , elongation , termination Catalyzed by RNA polymerase. Transcription.

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slide1

Genetic Information Flow: Transcription of Class I, II, III Genes

YILDIRIM BEYAZIT UNIVERSITY FACULTY OF MEDICINE

THE DEPARTMENT OF MEDICAL BIOLOGY

ASST. PROF. DR. ENDER ŞİMŞEK

transcription
Transcription
  • Initiation, elongation, termination
  • Catalyzed by RNA polymerase
transcription1
Transcription
  • “Transcription bubble”: DNA transiently separated into single strands
  • Only one strand is used as a template
  • Unwinding point & rewinding point
slide4
OVERVIEW OF TRANSCRIPTION
  • The first stage in the expression of genetic information is transcription.
slide5
OVERVIEW OF TRANSCRIPTION
  • The first stage in the expression of genetic information is transcription.
  • For any particular gene, only one strand of the DNA molecule(the template strand) is copied by RNA polymerase.
slide6
OVERVIEW OF TRANSCRIPTION
  • RNA polymerase moves along the template strand in the 3' to 5' direction as it synthesizes the RNA product in the 5' to 3' direction using NTPs (ATP, GTP, CTP, UTP) as substrates.
slide7
OVERVIEW OF TRANSCRIPTION
  • RNA polymerase moves along the template strand in the 3' to 5' direction as it synthesizes the RNA product in the 5' to 3' direction using NTPs (ATP, GTP, CTP, UTP) as substrates.
  • The coding (non-template) strand is not used during transcription. It is identical in sequence to the RNA molecule, except that RNA contains uracil instead of the thymine found in DNA.
slide8
TRANSCRIPTION:

RNA polymerase locates genes in DNA by searching for promoter regions.

The promoter is the binding site for transcription factors and RNA polymerase.

Bindingestablishes where transcription begins and in which direction transcription proceeds.

slide10
In a gene, a numbering system is used to identify the location of important bases. The first base transcribed as RNA is defined as the + 1 base of that gene region.
slide11
In a gene, a numbering system is used to identify the location of important bases. The first base transcribed as RNA is defined as the + 1 base of that gene region. To the left (5', or upstream) of this starting point for transcription, bases are -1, -2, -3, etc. to the right (3', or downstream) of this point, bases are +2, +3, etc.
slide13

Prokaryotic RNA Polymerase

  • There is a single prokaryotic RNA polymerase that synthesizes all types of RNA in the cell.
slide14

Prokaryotic RNA Polymerase

  • There is a single prokaryotic RNA polymerase that synthesizes all types of RNA in the cell.
  • The core polymerase responsible for making the RNA molecule has the subunit structure (ααββ`).
slide15

The transcription

Steps of Core & Sigma:

DNA binding

Promoter search

Closed promoter

complex formation

Open promoter

complex formation

slide17

A protein factor calledsigma (σ) is required for the initiation of transcription at a promoter.

slide24
The following events occur

during the expression of a prokaryotic gene:

  • With the help of sigma factor, RNA polymerase recognizes and binds to the promoterregion.
    • The bacterial promoter contains two "consensus" sequences, called the Pribnow box (or TATA box) and the -35 sequence. The promoter identifies the start site for transcription and orients the enzyme on the template strand.
slide25
The following events occur

during the expression of a prokaryotic gene:

2. Transcription begins at the + 1 base pair. Sigma factor is released as soon as transcription is initiated.

slide26
The following events occur

during the expression of a prokaryotic gene:

3. The core polymerase continues moving along the template strand in the 3' to 5' direction, synthesizing the mRNA in the 5' to 3' direction.

slide27
The following events occur

during the expression of a prokaryotic gene:

4. RNA polymerase eventually reaches a transcription termination signal, at which point it will stop transcription and release the completed mRNA molecule.

slide28
There are two kinds of transcription terminators commonly found in prokaryotic genes:
  • Rho-independent termination occurs when the newly formed RNA folds back on itself to form a GC-rich hairpin loop closely followed by 6-8 U residues. These two structural features of the newly synthesized RNA promote dissociation of the RNA from the DNA template.
slide32
There are two kinds of transcription terminators commonly found in prokaryotic genes:
  • Rho-dependent termination requires participation of rho factor. The rho protein binds to the newly formed RNA and moves toward the RNA polymerase that has paused at a termination site. Rho then displaces RNA polymerase from the 3' end of the RNA.
slide36
The following events occur

during the expression of a prokaryotic gene:

5. Transcription and translation can occur simultaneously in bacteria. Because there is no processing of prokaryotic mRNA (generally no introns), ribosomes can begin translating the message even before transcription is complete.

slide37
The following events occur

during the expression of a prokaryotic gene:

6. The ribosome translates the message in the 5' to 3' direction, synthesizing the protein from amino terminus to carboxyl terminus.

slide38
A Prokaryotic Transcription Unit
  • Prokaryotic Polycistronic Message Codes for Several Different Proteins

In these cases, related genes grouped together in the DNA are transcribed as one unit. (The mRNA in this case contains information from several genes and codes for several different proteins.)

slide40
There are three eukaryotic RNA polymerases, distinguished by the particular types of RNA, they produce:
  • RNA polymerase I (Class I gene) is located in the nucleolus and synthesizes 28S, 18S, and 5.8S rRNAs.
slide41
There are three eukaryotic RNA polymerases, distinguished by the particular types of RNA, they produce:
  • RNA polymerase I (Class I gene) is located in the nucleolus and synthesizes 28S, 18S, and 5.8S rRNAs.
  • RNA polymerase II (Class II gene) is located in the nucleoplasm and synthesizes hnRNA/mRNA and some snRNA.
slide42
There are three eukaryotic RNA polymerases, distinguished by the particular types of RNA, they produce:
  • RNA polymerase I (Class I gene) is located in the nucleolus and synthesizes 28S, 18S, and 5.8S rRNAs.
  • RNA polymerase II (Class II gene) is located in the nucleoplasm and synthesizes hnRNA/mRNA and some snRNA.
  • RNA polymerase III (Class III gene) is located in the nucleoplasm and synthesizes tRNA, some snRNA, and 5S rRNA.
initiation
INITIATION:
  • The core of RNA pol II enzyme can not recognize the promoter itself, it requires general transcription factors (GTFs).
initiation1
INITIATION:
  • The core of RNA pol II enzyme can not recognize the promoter itself, it requires general transcription factors (GTFs).
  • These proteins, however, do not take part in the RNA synthesis but their binding to the promoter forms an attraction for RNA polymerase.
  • GTFs: TFIIA, TFIIB, etc.
slide48
Eukaryotic promoter is located in the 5’ end of the transcription starting point.
  • TATA box of the promoter from the start of transcription (+1) is ahead of about 25-30 nucleotides upstream and has the transcription start point.
slide49
Eukaryotic promoter is located in the 5’ end of the transcription starting point.
  • TATA box of the promoter from the start of transcription (+1) is ahead of about 25-30 nucleotides upstream and has the transcription start point.
  • The region is called as TATA box is located in the region -30 and Transcription Binding Proteins (TBPs) bind to this region.
slide51
The TATA box is firstly recognized by transcription factor TFIID, after that TFIIB binds there.
  • TBP is a part of TFIID and after TBP binds to the promoter, the core of RNA pol II with GTFs form pre-initiation complex (PIC), which is composed of 6 GTFs.
slide52
The TATA box is firstly recognized by transcription factor TFIID, after that TFIIB binds there.
  • TBP is a part of TFIID and after TBP binds to the promoter, the core of RNA pol II with GTFs form pre-initiation complex (PIC), which is composed of 6 GTFs.
  • After the initiation, many GTFs leaves the complex and the primary transcript is synthesized.
slide53
Meanwhile, the other TFs (TFIIF, TFIIH) begin to accumulate on RNA pol.
  • TFIIH uses the energy of ATP hydrolysis to break DNA strand from the starting point of transcription.

***After the beta subunit of RNA pol II enzyme (carboxy tail domain-CTD) is phosphorylated by TFIIH the initiation ceases and elongation initiation starts.

termination
TERMINATION:

While the Eukaryotic RNA is synthesized, it is modified.

slide55

PRODUCTION OF EUKARYOTIC MESSENGER RNA

  • In eukaryotes, most genes are composed of coding segments (exons) interrupted by noncoding segments (introns).
  • Both exons and introns are transcribed in the nucleus.
    • Introns are removed during the processing of RNA molecule in the nucleus.
  • In eukaryotes, all mRNA is monocistronic. The mature mRNA is translated in the cytoplasm.
slide56
Transcription of a typical eukaryotic gene

occurs as follows:

1. With the help of transcription factors, RNA polymerase II recognizes and binds to the promoter region. The basal promoter region of eukaryotic genes usually has two consensus sequences called the TATA box (also called Hogness box) and the CAAT box.

slide57
Transcription of a typical eukaryotic gene

occurs as follows:

2. RNA polymerase II separates the strands of the DNA over a short region to initiate transcription and read the DNA sequence. The template strand is read in the 3' to 5' direction as the RNA product (the primary transcript) is synthesized in the 5' to 3' direction. Both exons and introns are transcribed.

processing of eukaryotic messenger rna
Processing of Eukaryotic Messenger RNA

The primary transcript must undergo extensive posttranscriptional processing inside the nucleus to form the mature mRNA molecule.

processing of eukaryotic messenger rna1
Processing of Eukaryotic Messenger RNA

The primary transcript must undergo extensive posttranscriptional processing inside the nucleus to form the mature mRNA molecule.

These processing steps include:

1. A 7-methylguanosine cap is added to the 5' end while the RNA molecule is still being synthesized. The cap structure serves as a ribosome-binding site and also helps to protect the mRNA chain from degradation.

slide61
2. A poly-A tail is attached to the 3' end. In this process, an endonuclease cuts the molecule on the 3' side of the sequence AAUAAA (poly-A addition signal), then poly-A polymerase adds the poly-A tail (about 200 A’s) to the new 3' end. The poly-A tail protects the message against rapid degradation and aids in its transport to the cytoplasm.
slide62
3. Introns are removed from hnRNA by splicing, accomplished by spliceosomes, which are complexes of snRNA and protein. The hnRNA molecule is cut at splice sites at the 5' (donor) and 3' (acceptor) ends of the intron.
slide63
3. Introns are removed from hnRNA by splicing, accomplished by spliceosomes, which are complexes of snRNA and protein. The hnRNA molecule is cut at splice sites at the 5' (donor) and 3' (acceptor) ends of the intron.

- The intron is excised in the form of a lariat structure and degraded. Neighboring exons are joined together to assemble the coding region of the mature mRNA.

slide64
4. All of the intermediates in this processing pathway are collectively known as hnRNA.

5. The mature mRNA molecule is transported to the cytoplasm, where it is translated to form a protein.

slide66
PRODUCTION OF OTHER CLASSES OF RNA
  • Genes encoding other classes of RNA are also expressed. The RNA products are not translated to produce proteins, but rather serve different roles in the process of translation.
slide67
PRODUCTION OF OTHER CLASSES OF RNA
  • Genes encoding other classes of RNA are also expressed. The RNA products are not translated to produce proteins, but rather serve different roles in the process of translation.

RIBOSOMAL RNA (rRNA) IS USED TO CONSTRUCT RIBOSOMES

  • Eukaryotic ribosomal RNA is transcribed in the nucleolus by RNA polymerase I as a single piece of 45S RNA, which is subsequently cleaved to yield 28S rRNA, 18S rRNA, and 5.8S rRNA.
slide68
PRODUCTION OF OTHER CLASSES OF RNA
  • Genes encoding other classes of RNA are also expressed. The RNA products are not translated to produce proteins, but rather serve different roles in the process of translation.

RIBOSOMAL RNA (rRNA) IS USED TO CONSTRUCT RIBOSOMES

  • Eukaryotic ribosomal RNA is transcribed in the nucleolus by RNA polymerase I as a single piece of 45S RNA, which is subsequently cleaved to yield 28S rRNA, 18S rRNA, and 5.8S rRNA.
  • RNA polymerase III transcribes the 5S rRNA unit from a separate gene. The ribosomal subunits assemble in the nucleolus as the rRNA pieces combine with ribosomal proteins.
transfer rna trna carries activated amino acids for translation
Transfer RNA (tRNA) Carries Activated Amino Acids for Translation
  • There are many different specific tRNAs. Each tRNA carries only one type of activated amino acid for making proteins during translation.
transfer rna trna carries activated amino acids for translation1
Transfer RNA (tRNA) Carries Activated Amino Acids for Translation
  • There are many different specific tRNAs. Each tRNA carries only one type of activated amino acid for making proteins during translation.
  • The genes encoding these tRNAs in eukaryotic cells are transcribed by RNA polymerase III.
  • The tRNAs enter the cytoplasm where they combine with their appropriate amino acids.