RNA expression and processing

What are genes the templates for?

▪ Genes are templates for the synthesis of an RNA molecule

▪ All genes are written in DNA and encode an RNA product

▪ Transcription: RNA polymerase uses a DNA template to guide the synthesis of a complementary RNA

Gene expression

is the process of synthesizing the product encoded by a gene, and begins with transcription

RNA processing

After they are transcribed, RNAs must be processed

▪ Newly made RNA is called a primary transcript

▪ Most primary transcripts undergo RNA processing

▪ rRNA must be to produce mature functional RNA

▪ Some RNAs are edited

▪ RNA polymerase II transcripts undergo extensive RNA processing to make a mature mRNA

▪ 5’ cap, 3’ polyadenylation, splicing

The Abundance of mRNA Allows what?

The Abundance of mRNA Allows Amplification of Genetic Information

▪ Human cells have two copies of most genes

▪ Individual genes can be transcribed repeatedly

▪ E.g., Two genes → hundreds of transcripts per cell

▪ This allows for amplification of genetic information

mRNA stability

▪ mRNA is unstable compared to DNA

▪ Most are degraded within a few hours

▪ Transcription, mRNA processing, mRNA stability, and translation are all regulated by the cell to control gene expression

What do genes require to be trascribed?

A gene requires a promoter to be transcribed

RNA polymerase is the multiprotein complex that catalyzes transcription

▪ Promoters are DNA sequences at the 5’ ends of genes

▪ Sequences in promoters are bound by proteins, which recruit RNA polymerase

▪ General transcription factors are proteins that bind promoter sequences and recruit RNA polymerase are called

Role of General Transcription Factors in Binding RNA Polymerase II to DNA.

This figure outlines the sequential binding of six general transcription factors (called TFII_, where _ is a letter identifying the particular factor) and RNA polymerase

a. TFIID binds to TATA (core promoter-minimal set of DNA sequences sufficient to direct the accurate initiation of transcription by RNA polymerase.) box in DNA

b. TFIIA and TFIIB form complex with TFIID

c.Resulting complex is bound by RNA polymerase attached to TFIIF

Eukaryotic RNA polymerases

RNA pol I rRNA

RNA pol II mRNA (and some other RNAs)

RNA pol III tRNA (and other small RNAs)

General transcription factors

▪ General transcription factors (GTF): proteins that bind core promoters of all nuclear genes

▪ GTFs bind promoters in sequential order

▪ Each RNA polymerase has its own GTFs

▪ Nomenclature: TFIID is a GTF for RNA pol II

▪ D indicates a specific protein complex

Transcription initiation

▪ TFIID – binds DNA sequences specifically found in promoters

▪ TFIIH – last to arrive; has 2 jobs: Unwind DNA, Phosphorylate RNA pol II

▪ Phosphorylation is the “Go” signal for transcription initiation

Explination of the figure that outlines the sequential binding of six general transcription factors (called TFII_, where _ is a letter identifying the particular factor) and RNA polymerase:

1. TFIID binds to TATA box in DNA

2. TFIIA and TFIIB form complex with TFIID

3. Resulting complex is bound by RNA polymerase attached to TFIIF

4. Preinitiation complex is completed by addition of TFIIE and TFIIH

5. Rna polymerase CTD undergoes phosphorylation

6. In intact chromatin, the efficient binding of general transcription factors and RNA polymerase to DNA requires the participation of additional regulatory proteins that open up chromatin structure and facilitate assembly of the preinitiation complex at specific genes.

Transcription of DNA occurs in four main stages

➊ binding of RNA polymerase to DNA at a promoter, ➋ initiation of transcription on the template DNA strand, ➌ subsequent elongation of the RNA chain, and ➍ eventual termination of transcription, accompanied by the release of RNA polymerase and the completed RNA product from the DNA template. RNA polymerase moves along the template strand of the DNA in the direction, and the RNA molecule grows in the 5→3 direction.

Key steps of transcription

Before transcription can begin, RNA pol must bind the promoter and unwind DNA Key steps of transcription

1. Initiation

2. Elongation

3. Termination These steps are required for transcription of all genes in all species

Transcription elongation

▪ Nucleosomes must be disassembled for RNA polymerase to access DNA

▪ Nucleosomes must be immediately reassembled after the enzyme passes

▪ Requires chromatin remodeling proteins

▪ Elongation through the DMD gene (2.3 million base pairs) takes ~16 hours

Transcription elongation and termination

▪ RNA pol II termination is often coupled to polyadenylation

▪ Primary transcript cleaved ~10-35 nucleotides after polyadenylation signal

▪ The cleavage site is also the site for addition of the poly(A) tail © 2016 & 2021

▪ ~50-250 adenosines attached to mRNA after transcription

▪ Poly(A) tails stabilize mRNA and play a role in translation

mRNA splicing

▪ Not all sequences in the primary transcript are included in the mRNA

▪ “Expressed” sequences are called exons

▪ The other “intragenic” sequences that are removed are called introns ▪ Exons must be spliced together after introns are discarded

Alternative Splicing

▪ Introns allow alternative splicing of pre-mRNA

▪ Certain splice sites can be activated or skipped

▪ Allows one gene to encode multiple polypeptides

▪ ~Half of human genes undergo alternative splicing

▪ Some human genes have hundreds of splice variants

What is a 5’ cap?

mRNA made by RNA polymerase II have 5’ caps

▪ 5’ ends of mRNAs have 5’ caps

▪ Modified guanosine with a “backward” linkage to the mRNA (7methylguanosine)

▪ Added soon after transcription is initiated

▪ 5’ cap stabilizes mRNA and is important for translation

▪ Ribosomes assemble at 5’ caps and then scan mRNA until they find start codons (5’-AUG-3’)

The RNA Pol II Coordinates what?

The RNA Pol II Coordinates RNA Processing

▪ Many RNA processing events occur during transcription

▪ The long C-terminal domain (CTD) of RNA polymerase recruits RNA processing proteins to the site of transcription

▪ The CTD binds enzymes needed for capping, splicing, and cleavage/polyadenlylation

Review of protein coding genes

• Introns are usually much larger than exons

• Exon numbers vary from gene-to-gene

• Some exons undergo alternative splicing

• Selectively included or excluded from the final transcript

RNA Editing

RNA Editing Allows Sequences to Be Altered

▪ Another type of RNA processing is RNA editing

▪ Insertion, removal, or alteration of nucleotides in RNA

▪ Frequent in tRNA

▪ Also occurs in some brain mRNAs

▪ Alteration of nucleotides includes conversion to “modified” bases

▪ E.g., Adenosine converted to inosine ▪ Inosine can form base pairs with cytosine, adenine, or uracil

Processing and Secondary Structure of Transfer RNA.

• Every tRNA gene is transcribed as a precursor that must be processed into a mature tRNA molecule.

• processing for this tRNA involves removal of the leader sequence at the 5′ end, replacement of two nucleotides at the 3′ end by the sequence CCA (which serves as an attachment site for amino acids in all mature tRNAs), chemical modification of certain bases, and excision of an intron. T

• The mature tRNA is depicted in a flattened cloverleaf representation, which clearly shows the base pairing between self-complementary stretches in the molecule.

Processing of ribosomal RNA

▪ Ribosomes are made of protein and ribosomal RNA (rRNA)

▪ rRNA: most abundant and stable RNA in eukaryotic cells

▪ Four types of rRNA, named for sedimentation rates during centrifugation (S – Svedberg unit; ~correlates with size)

Four types of rRNA:

• Large (60S),Coefficient 25–28S, 4700 Nucleotides

• Large (60S),Coefficient 5.8S, 160 Nucleotides

• Large (60S), ,Coefficient 5S, 120 Nucleotides

• Small (40S), Coefficient 18S, 1900 Nucleotides