Genetics Chapter 8

RNA Nucleotides and Structure

Single-Stranded structure can lead them to adopt folded secondary structures
Two cricital chemical differences in comparison to DNA
1) The second pyrimidine is uracil rather than thymine.
2) The sugar ribose rather thand deoxyribose.

Ribonucleotides

Composed of ribose, one or more phosphate groups, and one of four nitrogenous bases, the nucleotides that make up RNA.

Uracil

One of the two types of pyrimidine nucleotides in RNA. Thymine has a methyl group at the 5 position of the pyrimidine ring, whereas uracil does not.

RNA polymerase

The enzyme that catalyzes the synthesis of RNA. Addition at the 3' end of the nascent strand.
A single type of E. coli RNA polymerase catalyzes transcription of all RNAs.
Bacterial RNA polymerase is composed of a pentameric core enzyme that binds to a six

Sydney Brenner, Fancois Jacob, and Matthew Meselson

Identified an unstable form of RNA as the genetic messenger.
Designed an experiment using bacteriophage T2 and E. coli to investigate whether phage protein synthesis requires newly constructed ribosomes, or whether phage proteins could be produced using e

Messenger RNA

Form of RNA transcribed from a gene and subsequently translated to produce a polypeptide or protein.

Transfer RNA

A family of small RNA molecules that each bind a specific amino acid and convey it to the ribosome, where the anticodon sequence undertakes complementary basepairing with an mRNA codon during translation.

Ribosomal RNA

A group of RNA molecules that compose part of the structure of ribosomes.

Micro RNA (miRNA)

Small (21-24 nuts) regulatory RNAs produced by Dicer and acting in a RISC complex to either repress translational or cleave target mRNA molecules.
Important role in the post transcriptional regulation of mRNA, regulating protein production through a proce

Small interfering RNA (siRNA)

Help protect plant and animal genomes from the production of viruses and from the spread of transposable genetic elements within the genome.
Single-stranded 21-24 nucleotide RNA molecules derived from either endogenous or exogenous double stranded RNA mol

Ribozymes

Catalytically active RNAs.

Template strand

The DNA strand serving as a template for synthesis of a complementary nucleic acid strand.

Coding Strand

The nontemplate strand of DNA that has the same 5'-to'3' polarity as its transcript and the same sequence, except for T in DNA and U in RNA.

Promoter

A regulatory sequence of DNA near the 5' end of a gene that acts as the binding location of RNA polymerase and directs RNA polymerase to the start of transcription.
Immediately upstream.

Upstream

Referring to a gene or sequence location that is towards the 5' direction of a coding strand. Example: the promoter.

Termination Region

Regulates the cessation of transcription. Immediately downstream.

Four Stages of Transcription (E. coli)

1) Promoter Recognition
2) Chain Initiation
3) Chain Elongation
4) Chain Termination

Core Enzyme (E. coli)

The five-polypeptide component of bacterial RNA polymerase that actively carries out transcription.
By itself the core enzyme can transcribe DNA template-strand sequence into RNA sequence, but the core enzyme is unable to bind to a promotor or initiate RN

Sigma Subunit (E. coli)

Accessory protein that changes the promoter-recognition specificity of the bacterial RNA polymerase core enzyme.
The core enzyme is unable to bind to a promotor or initiate RNA synthesis without a sigma subunit.
Causes a conformational shift that enables

Bacterial promoters

Double-stranded DNA sequence that is the binding site for RNA polymerase. Regulate transcription since RNA polymerase cannot initiate transcription without binding to promoter sequence.
Upstream of the coding sequence.
RNA polmerase is attracted to promot

Consensus Sequences

A nucleotide sequence in a DNA segment derived by comparing sequences of similar segments from other genes or organisms. THe most commonly occurring nucleotides at each position comprise the sequence.

Transcription Initiation (prokaryotes)

RNA polymerase holoenzyme initiates through a 2 step process.
1) makes an initial loose attachment to the double stranded promoter sequence and then binds tightly to form the "closed promoter complex"
2) The bound holoenzyme unqinds approximately 18 bp or

Transcription Elongation (prokaryotes)

Upon reaching the +1 nucleotide, the holoenzyme begins RNA synthesis by using the template strand to direct RNA assembly. Around the 8-10 the RNA nucleotide the sigma subunit dissociates from the core enzyme.
40 nucleotides / second
DNA unwinds.
When tran

Intrinsic Termination

In bacterial transcription, the DNA sequence-dependent mechanism for transcription termination.
Inverted repeat DNA sequences induce formation of 3' mRNA stem-loop (hairpin) structures that are followed by multiple uracils.
Characterized by a string of ad

Rho-dependent Termination

The process of bacterial transcription termination involving rho protein.
Less frequent than intrinsic termination.
Characterized by a different terminator sequence, "rho utilization site" or rut site.
Rho protein is composed of 6 identical polypeptides.

Two transcription mechanisms that occur in bacteria

1) Intrinsic Termination
2) Rho-dependent Termination

rho utilization site

The site of attachment of rho protein that aids in rho-protein-driven bacterial transcription termination.
A stretch of ~50 nucleotides that is rich in cytosine and poor in guanine.

Eukaryotic Transcription differs

1) "E" promoters and consensus sequences are considerably more diverse than in E. coli
2) "E" have three different promoters, transcribe, & produce different RNAs.
3) In "E" the molecular apparatus is more complex.
4) "E" genes contain introns and exons,

Chromatin

The complex of nucleic acids and proteins that compose eukaryotic chromosomes.
Directly affects transcription and plays a central role in regulating eukaryotic transcription by way of changing the strength of protein association with DNA that can permit o

Eukaryotic Polymerases

RNA pol I - Transcribes three rRNA genes
RNA pol II - Responsible for mRNAs and small nuclear RNA genes
RNA pol III - tRNA genes and one small nuclear RNA gene and one rRNA gene

Consensus Sequences for RNA Polymerase II Transcription

RNA pol II transcribes mRNA.
Promoters are numerous and highly diverse.
Each play important roles in the binding of transcription factors.

Researchers locate promoter DNA (eukaryotes) *I don't know if i should include this.

(1) Determining which DNA sequences are bound by protein associated with RNA pol II turing transcription
(2) Putative promoter sequences from different genes are compared to evaluate their similarities
(3) Mutations that alder gene transcription are exami

TATA box (or Goldeberg-Hogness box)

The thymine- and adenine-rich consensus sequence region found in most eukaryotic promoters.
6bp, TATAAA
Locates -25 relative to the transcriptional start site.

CAAT box

A common consensus sequence component of eukaryotic promoters.
Commonly located near -80
4bp

GC Rich Box

An occasional upstream consensus sequence of eukaryotic promoters that is rich in guanine and cytosine.
Commonly located near -90 or more upstream
GGGCGG

Transcription Factors (TF)

Proteins that bind promoters and are functional in transcription.

Initial Committed Complex

In eukaryotic transcription, a partially completed mulitprotein complex that is preparing to bind RNA polymerase II.
Example: TATA-binding protein (TBP) binds the TATA box and TBP-associated factor (TAF).

Minimal Initiation Complex

In eukaryotic transcription, a partially completed mulitprotein complex that is preparing to bind RNA pol II.
Example: TFIIB, TFIIF, and RNA pol II join the initial committed complex.

Complete Initiation Complex

The multi-subunit complex that forms at the promoter immediately before the onset of transcription.
Contains "General Transcription Factors"
Once assembled, this directs RNA pol II to the +1 nucleotide on the template strand, where it begins the assembly

Detecting Promoter Consensus Elements

1) Discovering the presence and location of DNA sequences that transcription factor proteins will bind to.
2) Multational analysis to confer the functionality of the sequence. - Produce many different point mutations and compare level of transcription.
..

Enhancers Sequences

Sets of regulatory sequences that bind specific transcriptional proteins that can elevate transcription of targeted eukaryotic genes.
Often located upstream, but can be located downstream.Bind activator proteins to form a protein bridge with a bend. Incre

Silencer Sequences

Regulatory DNA sequences that can repress transcription of specific genes that may be located distantly from the sequence.
Also induce bens, but reduce transcription of targeted genes.

Transcription Factor Availability

Dependent on the synthesis of the transcription factors themselves and on their availability in the cell.
TF synthesis can be tightly regulated.
The availability of activated
Transcription factors is controlled through signal-transduction pathways.
Once a

RNA Pol I Promoters

(Remember RNA pol I is the most specialized RNA polymerase.
It transcribes ribosomal RNA genes found in the nucleolus.)
Promoters recognized by RNA pol I contain 2 functional sequences:
(1) Core element - Essential for transcription initiation.
(2) Upstre

RNA Pol III Promoters

(Remember RNA pol III is primarly presponsible for transcription of tRNA genes)
Small nuclear RNA genes have three upstream elements, whereas the genes for 5S ribosomal RNA and transfer RNA each contain two internal promoter elements that are downstream o

Termination in RNA Pol III

Similar to E. coli.
RNA pol III transcribes a terminator sequence that contains a string of uracils.
The RNA pol III does not contain an inverted repeat, so no stem-loop structure forms near the 3' end of RNA

Termination in RNA Pol I

Terminated at a 17 bp consensus sequence that binds transcription-terminating factor I (TTFI) - a specific protein that binds a termination sequence to stop transcription.

Post-Transcriptional Processesing

Pre-mRNA" is modified into "Mature mRNA" which migrates out of the nucleus to the cytoplasm for translation.
Modification Steps:
(1) 5' Capping
(2) 3' Polyadenylation
(3) Intron Splicing
These processes are controlled by proteins and are closely coupled

5' Capping

In eukaryotic pre-mRNA processing, the addition of 7-methylguanosine to the nucleotide at the 5' end of pre-mRNA by a triphosphate bridge. Methylation of adjacent nucleotides may also occur.
Guanylyl Transferase adds guanine to the 5' end of the pre-mRNA

3' Polyadenylation

During eukaryotic pre-mRNA processing, and enzyme-driven modification that removes the 3' end of the pre-mRNA and adds numerous adenines to form the poly-A tail.
(1) Binding of the cleavage and polyadenylation specificity factor (CPSF) near the polyadenyl

Tailless mRNA

The 3' end contains a short stem-loop structure reminiscent of the ones seen in the intrinsic transcription termination of bacteria.
Example: transcripts of genes producing histone proteins

Intron Splicing

The spliceosome complex-driven process that removes introns from eukaryotic pre-mRNA and ligates exonts to form mature mRNA.
There is a consensus sequence near each intron end to assist in accurate identification (5' and 3' splice sites and branch site).

Richard Roberts and Phillip SHarp

Discovered "split genes" in the eukaryotic genome.
R-looping:
DNA encoding a gene is isolated, denatured to single stranded form, and then mixed with mature mRNA transcript from the gene. Hybridiziation showed single stranded looping sections between matu

SR Proteins

Proteins rich in serene and arginine; operative in intron splicing.

Is there an order which introns are removed from pre-mRNA

Introns appear to be removed one by one, but not necessarily in order along the pre-mRNA

Carboxyl Terminal Domain (CTD)

Associates with proteins that carry out capping (CAP), intron splicing (SF), and polyadenylation (pA) so that the processes of transcription and pre-mRNA proceessing occur simultaneously.
Located at the site of emergence of mRNA from the polymerase.
mRNA

Alternative pre-mRNA processing

Difference between # of genes and # of polypeptides. It is common for large eukaryotic genomes to express more proteins than there are genes.
(1) Pre-mRNA can be spliced in alternative patterns in different types of cells
(2) alternative promoters can ini

Alternative intron splicing

Pre-mRNA can be spliced in alternative patterns in different types of cells.
Common in mammals but is less common in other animals and is rare in plants.
Largely controlled by variation of SR proteins in different types of cells.

Intron Self-Splicing

The capacity of certain RNA transcripts to undergo self-generated splicing that does not require splicing enzymes of the splisosome complex.

rRNA Processing

rRNA are transcribed as large precursor molecules that are cleaved into smaller RNA molecules by removal and discarding of spacer sequences. After processing the rRNAs fold into complex secondary structures and are joined by proteins to form ribosomal sub

tRNA processing

Different between bacteria and eukaryotes.
Bacteria- Some are transcribed as part of a large pre-tRNA transcript that is then cleaved to yield multiple tRNA molecules & some are produced similar to rRNA described.
Eukaryotes: tRNA genes occur in clusters

Third Base Wobble

The flexibility of purine-pyrimidine base pairing between the third base of a codon and the corresponding nucleotide of the anticodon.

RNA editing

The process of post-transcriptional addition or removal of nucleotides of certain mRNAs.
Two Types:
(1) guide RNA
(2) base substitution