BIO111 - Molecular genetics - Keywords

Eukaryotic promoters (Eukaryotic pol II promoters) consist of a core promoter, to which RNA pol II binds, and which overlaps with the transcription start site, promoter proximal elements (regulatory promoter), situated right upstream of the core promoter and enhancers and other regulatory elements, which can be located very far away from the core promoter. 

The most common consensus sequence of the core promoter is the TATA box, located around -25 to -30 bp, which is bound by the TATA box binding protein (TBP). There might be other consensus sequences present in addition, or instead of, the TATA box. 

All of the consensus sequences are bound by conserved basal transcription factors which then recruit assembly of the RNA pol II holoenzyme. 

Located just upstream of the core promoter, it can contain binding sites for many common transcription factors. Most of the factors that bind to the upstream promoter elements enhance transcription, other can reduce it. 

Normally, RNA pol in prokaryotes stops after it has transcribed through a terminator. In bacteria, there are two types of terminators: 

- Rho-independent termination

- Rho-dependent termination

Rho-independent terminators have two factors: 

- Inverted repeats (or palindromes) that can form a hairpin structure, once transcribed into RNA

- A poly-A stretch on the templante strand (resulting in a stretch of Us in the RNA)

1. Hairpins can cause RNA pol to slow down or even to stall. 

2. The A/U DNA/RNA hybrid base pairs result in a weak association of the RNA with the DNA, allowing the RNA to fall of the template. 

3. Once the RNA has dissociated from the template, RNA pol falls off. 

Rho-dependent terminators require the rho helicase for termination of transcription. They have two factors: 

- Inverted repeats (or palindromes) that can form a hairpin structure once transcribed into RNA

- A stretch of DNA upstream of the inverted repeats that forms no secondary structure in the RNA. 

1. Hairpins cause RNA pol to slow down or stall

2. The unstructured region of the RNA allows binding of the helicase rho

3. Rho moves along the RNA in a 5' to 3' fashion. If it catches up with RNA polymerase, it unwinds the RNA/DNA hybrid, thereby releasing the RNA

Upstream and downstream describe direction. 

5' direction = upstream

3' direction = downstream

As the RNA is not double stranded but single stranded, it can form special secondary structures. 

Because of base pairing, RNA chains can now fold themselves to form various structures: 

Synthesizes large rRNAs

Each core RNA polymerase consists of multiple protein subunits. 

Synthesizes mRNAs, some snRNAs, miRNA precursors 

Each core enzyme associates with many accessory proteins

Synthesizes 5s rRNA, tRNAs, some snRNAs and other small RNAs

Each RNA polymerase will bind to a different consensus promoter sequence

Protein that controls the rate of transcription of genetic information form DNA to mRNA by binding to a specific DNA sequence and promoting or supressing transcription. This helps to regulate the expression of genes near that sequence. 

Transcription factors contain at least one DNA-binding domain (DBD). 

Short region of DNA that can be bound by proteins to increase the likelyhood that the transcription of a particular gene will occur. They are part of the familiy of transcription factors. 

The TATA box is the most common consensus sequence in the eukaryotic promoters. TATA box binding protein (TBP) binds to it and recruits the RNA pol II and other transcription factors. 

It is located ca. -25 to -30 bp. 

RNA pol I: 

Uses a termination factor. This termination factor binds to a DNA sequence downstream of the termination site. 

RNA pol II: 

Stops at various places in a region that can stretch over several hundred base pairs. Termination is likely promoted once the mRNA has been cleaved and the polyA tail added. 

RNA pol III: 

Terminates after a string of Us. There is no need for an hairpin before the string of U residues.

TATA binding protein. Part of the TFIID (Transcription Factor II D). It binds to the TATA box, recruting RNA pol II and the other TFs to create a pre-initiation complex (PIC). 

After transcription has started, RNA pol II separates from most of the TFs, which remain at the promoter and can recruit the next RNA pol II

Process in which proteins or nucleic acids lose the quaternary, tertiary and secondary structure. This is done by application of some external stress or compound such as a strong acid or a base, an organic solvent, radiation, heat, etc. 

Denaturated proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggreagation due to the exposure of hydrophobic groups. 

The inverse process of denaturation. Is only possible in very few cases. This means that the protein in question can regain its native state when the denaturing influence is removed. 

Melting of DNA means that a dsDNA is put under a stress which causes the ds to break open and become ssDNA. The melting temperature of a dsDNA is defined as the temperature at which 50% of the DNA molecules are dissociate into single strands. 

Also called annealing. Process by which to single strands of DNA combine to form dsDNA. 

Process of annealing two ssDNA together. This is done by : 

1. Heating up of dsDNA (weakening of the H-Bonds between bp trough encreased entropy) (DNA melting or denaturation)

2. Cooling down of the solution (slowly) allows the two strands to base pair again. (Renaturation or reannealing)

A hybrid is a molecule of two ssDNA of nucleic acid from different origins that have complementary sequences and can therefore base pair with each other. 

It can be performed with two DNA strands, two RNA strands or with one RNA and one DNA strand: 

Exons are the parts of the gene that are found in the mature RNA. Exonic sequences can be protein coding or non-coding. 

Introns are internal sequences (sequences between exons) that are transcribed but not found in the mature RNA. They are most commonly found in eukaryotic genes coding for mRNAs. Introns are removed from the intial transcript by splicing. 

In cis (in the mRNA): 

5' splice site: intron always starts with GU 

3' splice site: intron always ends with AG 

Branch point: Always an A

Small nuclear ribonucleoprotein particels (snRNPs, "snurps"): componsed of one snRNA and several proteins: 

- U1

- U2

-U4

-U5

-U6

Process of removal of introns. Depends on the group on introns: 

1: In some rRNA genes, the type is self-splicing

2: In protein-encoding genes in mitochondria and chloplastes it is also self-splicing

3: In nuclear pre-mRNA, containing protein-encoding genes in the nucleus, it is doneby a spliceosomal

4: In tRNA containing tRNA genes it is done by enzymes. 

Splicing results in the fusion of two exons and the release of a lariat intron. 

The 5' cap consists of a 7-methyl Guanosine attached to the 5' -most nucleotide of an RNA trought an unusual 5' -P-P-P-5' bond. 

It promotes translation, prevents degradation by exonucleases, regulates nuclear export and promotes 5' proximal intron excision. 

The addition of the polyA tail (also called polyadenylation) is part of the process that produces mRNA for translation. The polyA tail consists of multiple AMP. 

Most eukaryotic mRNAs have this 50 - 250 nt. of Adenosines attached to the 3' end of their last exon. This tail is added by the polyA polymerases (PAP).

The polyA tail is important for nuclear export, translation and the stability of mRNA. It acts as the binding site for polyA-binding protein

Occurs soon after start of transcription. 

1. Capping enzymes recruited by the phosphorylated tail (CTD) of RNA pol II recognize the growing 5' end of the pre-mRNA

2. The gamma phosphate of residue 1 is removed

3. GTP is added to the 5' end of the pre-mRNA in a 5' - 5' bond, with release of pyrophosphate

4. Guanine base of cap is methylated on N7

5. 2' OH of residues 1 and 2 are possibly methylated. Bases of residues 1 and 2 are rarely also methylated

The pre-mRNA has an AAUAAA consensus sequence 10 - 30 nt upstream of the 3' cleavage site, and a U-rich sequence downstream of it. 

For the polyAdenylation, a large protein complex is needed that includes: 

- CPSF (cleavage and polyadenilation specificity factor)

- CstF (cleavage stimulation factor)

- Two cleavage factors (CFI, CFII)

- PolyA polymerase (PAP)

- Other polypeptides

1. CPSF binds to the AAUAAA sequence, CstF to the U-rich sequence, thus creating a loop

2. mRNA is cleaved between the two sequences (mechanism still unknown)

3. 3' sequences (still attached to the elongating RNA pol II) and bound factors are released

4. Cleaved off 3' end is degraded, RNA pol II gets a signal that it can stop transcribing

5. PolyA polymerase (template independent) adds ca. 10 As to the new 3' end

6. PolyA binding protein (PABII) binds to the growing polyA

7. PABII stimulates PAP to add further A residues

8. The whole polyA tail is ultimately covered by PABII

Small nuclear RNA. 

Is found in the nucleus and has the function of processing pre-mRNA by helping to cleave and modify rRNA and to assemble rRNAs into mature ribosomes.

Small nuclear ribonucleoprotein particles are composed of one snRNA and several proteins. 

A very important snRNP complex is called the spliceosome, which catalyzes pre-mRNA splicing. 

At the 3' splice site, the intron always ends with AG. It is one the three sequence areas important for splcing. 

The 3' splice marks the end of an intron and the beginning of a new exon.