(2024) Genetics Chapter 12 Gene transcription and RNA modification

What is a gene at the molecular level?

A segment of DNA used to make a functional product

Transcription

The first step in gene expression

- DNA is not altered

What three sequences are found in DNA?

- Regulatory sequence

- Promoter

- Terminator

Regulatory regions

DNA base sequences that define the beginning and end of a gene and regulate the level of RNA synthesis

Promoter

Site for RNA polymerase binding and signals the beginning of transcription

Terminator

Signals the end of transcription

What four sequences are found in mRNA?

- Ribosome-binding site

- Start codon

- Codons

- Stop codon

Ribosome-binding site

- Site for ribosome binding

- near where translation begins in mRNA

- In eukaryotes the ribosome scans the mRNA for a start codon

Start codon

Specifies the first amino acid in a polypeptide sequence

- Usually formylmethionine in bacteria

- Methionine in eukaryotes

Codons

3-nucleotide sequences within the mRNA that specify particular amino acids.

Stop codon

Specifies the end of polypeptide synthesis

Template strand

DNA strand that is actually transcribed

- RNA transcript is complementary to the template strand

Coding strand

Opposite DNA strand or the nontemplate strand

- Identical to the RNA transcript except for uracil instead of thymine

Transcription factors

Proteins that recognize the promoter and regulatory sequences of genes to control transcription

What direction does the template and nontemplate strands run?

Template strand runs 3' to 5'

Nontemplate strand runs 5' to 3'

What are the three steps of transcription?

Initiation

Elongation

Termination

Transcription initiation

The promoter functions as a recognition site for transcription factors. Transcription factors enables RNA polymerase to bind to the promoter. Following the binding, DNA is denatured into a bubble known as the open complex

Transcription elongation

RNA polymerase slides along the DNA in an open complex to synthesize RNA

Transcription termination

A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA

Structural genes

Over 90% of all genes

- Transcribed into mRNA and translated into polypeptides

Nonstructural genes

other 10%

- Never translated and the final products are RNA molecules

What is the promoter region in bacteria?

- Typically located "upstream" of the site where transcription of the gene actually begins

- (-35 to -10) sequence

TATAAT box (Pribnow)

The -10 sequence in the promoter region of bacterial DNA

RNA holoenzyme

RNA polymerase enzyme in E. coli that is composed of

- Core enzyme

- Sigma factor

Core enzyme of RNA polymerase

- five subunits (a2, B, B', w)

- Important in binding to the DNA and catalyzing RNA synthesis

Sigma factor of RNA polymerase

- one subunit (δ)

- Recognizes the promoter sequence

How does the RNA holoenzyme bind to DNA?

- Binds loosely to the DNA

- Scans the DNA until it finds the promoter (-35 to -10)

- The helix-turn-helix is involved in a tighter binding to the DNA

Closed complex

The beginning of initiation when the sigma factor binds to the promoter it forms the closed complex

Open complex

Formed when the TATAAT box in the -10 region is unwound due to the A-T only having two hydrogen bonds between them (easier to unwind)

When does initiation end in bacterial transciption?

After a short RNA strand is made in the open complex, the sigma factor releases and initiation ends

How does elongation continue after the sigma factor is released?

The core enzyme still left slides down the DNA to synthesize an RNA strand

How long is the open complex?

Only about 17 bases, behind the open complex, the DNA rewinds back into a double helix

What is the average rate of RNA synthesis?

43 nucleotides per second

What are the two mechanisms for termination in E.coli?

- Rho-dependent termination which requires a rho protein

-Rho-independent termination which does not require a rho protein

How does rho- dependent termination occur?

- A rho-utilization site (rut) exists in the mRNA

- The rho helicase protein binds to the rut site and moves towards the 3' end of the mRNA

- The RNA polymerase transcribes a region that forms a stem-loop and proceeds to the terminator site

- The stem loop causes the RNA polymerase to pause

- During the pause a rho protein catches up to the open complex and speerates the RNA-DNA hybrid

How does rho-independent termination occur?

Requires two things ...

1. Uracil rich sequence at the 3' end of the RNA

2. Stem-loop structure upstream of the uracil-rich sequence

- The stem loop causes RNA polymerase to pause and it is stabilized by NusA proteins

- The U rich regions are weak and the RNA polymerase spontaneously dissociates from the DNA

What are other terms for rho-dependent and rho-independent termination?

Rho dependent is EXTRINSIC

Rho independent is INTRINSIC

What three RNA polymerases aid in eukaryotic transcription?

- RNA polymerase 1

- RNA polymerase 2

- RNA polymerase 3

RNA polymerase 1

Transcribes all rRNA genes (except for the 5S rRNA)

RNA polymerase 2

Transcribes all protein encoding structural genes

RNA polymerase 3

Transcribes all tRNA genes, 5S rRNA gene, and microRNA genes

What are the components of RNA polymerases?

JAW - site of entry for DNA

RUDDER- Forces the RNA-DNA hybrid apart

CLAMP- Controls the movement of DNA through the enzyme

What do most eukaryotic promoter genes have?

1. Core promoter

- Typically a TATAAT box

- Transcriptional start site

2. Regulatory elements

- Enhancers or silencers

Basal transcription

The short sequence in the core promoter produces a low level of transcription

Describe regulatory elements and regulatory transcription factors

Regulatory elements are DNA sequences that affect the binding of RNA polymerase to the promoter

- Regulatory transcription factors bind to these elements and influence the rate of transcription

- Vary in location, but mostly found in the -50 to -100 region

What are two types of regulatory transcription factors?

ENHANCERS - stimulate transcription

SILENCERS - inhibit transcription

Factors that control gene expression can be divided into two types based on their action...

CIS-ACTING ELEMENTS

- DNA sequences that exert their effect only over a particular gene

- Ex. TATA box, enhancers, silencers

TRANS-ACTING ELEMENTS

- Regulatory proteins that bind to cis-acting elements

What three categories of proteins are required for basal transcription to occur at the promoter?

1. RNA polymerase 2

2. Five different proteins called the general transcription factors (GTFs)

3. A protein complex called the mediator

How does the assembly of transcription factors and RNA polymerase 2 at the TATA box lead to elongation?

- TFIID binds to the TATA box

- TFIIB binds to TFIID

- TFIIB promotes the binding of RNA polymerase 2 to the core promoter

- TFIIF binds to RNA polymerase 2

- TFIIE and TFIIH bind to RNA polymerase 2 to form the closed complex and the stage of preinitiation is complete

- TFIIH acts as a helicase to form an open complex and causes CTD phosphorylation

- CTD phosphorylation breaks the contact between the other proteins

- RNA polymerase now proceeds to the elongation stage

What forms the closed complex in the basal transcription apparatus?

TFIIE and TFIIH bound to the RNA polymerase 2

What is the mediator?

A large protein complex required for transcription.

- Mediates interactions between RNA polymerase 2 and various regulatory TFs that bind enhancers and silencers

How does the mediator play a pivotal role in the switch between transcription initiation and elongation?

It may phosphorylate the CTD and RNA polymerase 2 and it may regulate the ability of TFIIH to phosphorylate the CTD

How does RNA polymerase 2 termination occur in eukaroytes?

Pre mRNAs are modified by cleavage near their 3' end with subsequent attachment of a string of As

- Transcription terminates 500 to 2000 nucleotides downstream from the polyA signal

- Two models for termination, further research needed to determine which are correct

What is the colinearity of gene expression?

- The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA

- The sequence of codons in the mRNA provides the instructions for the sequence of amino acids in the polypeptide

Are eukaryotic structural genes always collinear with their functional mRNAs?

No, RNA modification of exons or coding sequences are interrupted by intervening sequences called introns

What happens to introns and exons in pre mRNA?

introns are removed or EXCISED

exons are connected or SPLICED

When does RNA splicing occur?

Commonly in eukaryotes and occasionally in bacteria

How are many nonstructural genes (like ribosomal RNAs) cleaved?

Many are initially transcribed into a large RNA and then enzymatically cleaved into smaller functional pieces

- This occurs in the nucleolus

How are transfer RNAs cleaved?

They are also made as large precursors, but are cleaved at both the 5' and 3' ends to make functional tRNAs.

What is the difference between exonucleases and endonucleases?

EXONUCLEASES

- Cleave a phosphodiester bond between two nucleotides at the end of one strand

ENDONUCLEASES

- Can cleave bonds within a strand

What is a ribozyme?

RNA that contains the catalytic activity

What are the three mechanisms for splicing?

1. Group 1 intron splicing

2. Group 2 intron splicing

3. Spliceosome

What is self splicing?

Splicing among group 1 and 2 introns

- Does not need enzymes

- The RNA functions as its own ribozyme

- Occurs mainly in rRNA and tRNA

Maturases

Proteins in vivo which enhance the rate of splicing

How does group 1 splicing occur?

A free guanosine binds to a site within the intron and breaks the bond between exon 1 and intron

- Results in exon 1 and 2 covalently joined

How does group 2 splicing occur?

2' hydroxyl from adenine within the intron breaks the bond between exon 1 and the intron

- Results in exon 1 and 2 linkage and the intron as a lariat

What is the spliceosome composed of?

Several subunits knowns as snRNPs

What do snRNPs do?

1. Bind to an intron sequence and recognize the intron-exon boundaries

2. Hold the pre-mRNA in the correct configuration

3. Catalyze the chemical reactions that remove introns and covalently link exons

What three sites are in the intron/exon boundaries and which ones are recognized by the spliceosome?

1. 5' splice site

2. branch site

3. 3' splice site

The 5' splice site and the branch sites are both recognised by the spliceosome

How does the spliceosome mechanism work?

- U1 snRNP binds to the 5' splice site

- U2 snRNP binds to the branch site

- U4/U6 and U5 act to loop and trim the intron out while the exon is brought closer together

- 5' splice site is cut

- lariat is formed

- U1 and U4 are released

- 3' splice site is cut

- exon 1 is connected to exon 2

- The intron in the form of a lariat is released with U2, U5, and U6 to later be degraded

What is alternative splicing?

A benefit of genes with introns where pre mRNA with multiple introns can be spliced in different ways to form different polypeptides all from a single gene

What percentage of genes in humans are alternatively spliced? and what is an example?

70%

Alternate splicing in tropomyosin pre-mRNA can lead to different forms of the protein that help cells contract differently

What is capping in eukaryotic mRNAs?

A process where 7-methylguanosines are covalently attached to the 5' end

- Occurs as the RNA polymerase 2 is synthesizing the pre-mRNA usually when the transcript is only 20-25 bases long

Describe the process of capping

- RNA 5' triphosphate removes a phosphate

- Guanylyltransferase hydrolyzes GTP

- GMP is attached to the 5' end and PPi is released

- Methyltransferase attaches to a methyl group

What is the purpose of the 7-methylguanosine cap?

The structure is recognized by cap-binding proteins

- Plays a role in movement

- Early stages of translation

- Splicing of introns

What is polyadenylation?

The addition of a string of adenine nucleotides at the RNA 3' end or a polyA tail

Is the polyA tail encoded in the gene sequence?

No, it is added enzymatically after the gene is completely transcribed

What is the polyadenylation signal sequence?

AAUAAA

Describe the process of polyadenylation?

- Endonuclease cleavage occurs about 20 nucleotides downstream from the polyadenylation signal sequence

- PolyA-polymerase adds adenine nucleotides to the 3' end