transcription and splicing

Genetics: Analysis and Principles

Robert J. Brooker

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

CHAPTER 12

GENE TRANSCRIPTION AND RNA MODIFICATION (processing)

Figure 12.112-5

The central dogma of genetics

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

Gene expression is the overall process by which the information within a gene is used to produce a functional product which can determine a trait in play with the environment


12.1 OVERVIEW OF TRANSCRIPTION

12-6

Figure 12.212-7

Signals the end of protein synthesis


The strand that is actually transcribed (used as the template) is termed the template strand

The opposite strand is called the coding strand or the sense strand The base sequence is identical to the RNA transcript

Except for the substitution of uracil in RNA for thymine in DNA

Transcription factors recognize the promoter and regulatory sequences to control transcription

mRNA sequences such as the ribosomal-binding site and codons direct translation

Gene Expression Requires Base Sequences

12-8


Transcription occurs in three stages Initiation Elongation Termination

These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA

sequences Transcription factors that control transcription bind

directly or indirectly to DNA

The Stages of Transcription

12-9

12-10Figure 12.3


Once they are made, RNA transcripts play different functional roles

Well over 90% of all genes are structural genes producing mRNA

The other RNA molecules are never translated: This collection appears much greater that initially believed; Some RNAs are 20-25 nts long that have important functions!

RNA Transcripts Have Different Functions

12-11


The RNA transcripts from nonstructural genes are not translated They do have various important cellular functions They can still confer traits

In some cases, the RNA transcript becomes part of a complex that contains protein subunits

For example Ribosomes Spliceosomes Signal recognition particles

RNA Transcripts Have Different Functions

12-12

Our molecular understanding of gene transcription came from studies involving bacteria and bacteriophages

Indeed, much of our knowledge comes from studies of a single bacterium E. coli, of course

In this section we will examine the three steps of transcription as they occur in bacteria


12.2 TRANSCRIPTION IN BACTERIA

12-14


Promoters are DNA sequences that “promote” gene expression: Events at this piece of DNA are needed to initiate RNA synthesis/transcription More precisely, they direct the exact location for the

initiation of transcription and determine when and how frequently a gene is transcribed.

Promoters are typically located just upstream of the site where transcription of a gene actually begins The bases in a promoter sequence are numbered in

relation to the transcription start site

Promoters

12-15

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-16

Figure 12.4 The conventional numbering system of promoters

Bases preceding this are numbered

in a negative direction

There is no base numbered 0

Bases to the right are numbered in a

positive direction

Most of the promoter region is labeled with negative numbers


Figure 12.4 The conventional numbering system of promoters

The promoter may span a large region, but specific short sequence elements are

particularly critical for promoter recognition and activity level

Sometimes termed the Pribnow box, after its

discoverer

Sequence elements that play a key role in transcription


Figure 12.5 Examples of –35 and –10 sequences within a variety of bacterial promoters

The most commonly occurring bases

For many bacterial genes, there is a good correlation between the rate of RNA

transcription and the degree of agreement with the consensus sequences


RNA polymerase is the enzyme that catalyzes the synthesis of RNA

In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme

Five subunits = 2’ Sigma factor

One subunit =

These subunits play distinct functional roles

Initiation of Bacterial Transcription

12-19


The RNA polymerase holoenzyme binds loosely to the DNA

It then scans along the DNA, until it encounters a promoter region When it does, the sigma factor recognizes both the –35

and –10 regions A region within the sigma factor that contains a helix-turn-helix

structure is involved in a tighter binding to the DNA

Refer to Figure 12.6

Initiation of Bacterial Transcription

12-20

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12-21Figure 12.6

Amino acids within the helices hydrogen

bond with bases in the promoter sequence

elements

12-23Figure 12.7

12-26

Similar to the synthesis of DNA

via DNA polymerase

Figure 12.8

On average, the rate of RNA synthesis is about 43 nucleotides per second!


Termination is the end of RNA synthesis It occurs when the short RNA-DNA hybrid of the open

complex is forced to separate This releases the newly made RNA as well as the RNA polymerase

E. coli has two different mechanisms for termination 1. rho-dependent termination

Requires a protein known as (rho) 2. rho-independent termination

Does not require

Termination of Bacterial Transcription

12-27

12-28

rho utilization site

-dependent terminationFigure 12.10

Rho protein is a helicase

12-29-dependent terminationFigure 12.10

12-30

-independent termination is facilitated by two sequences in the RNA 1. A uracil-rich sequence located at the 3’ end of the RNA 2. A stem-loop structure upstream of the Us

-independent terminationTermination in Eukaryotes is much less well defined !

Figure 12.11

URNA-ADNA hydrogen bonds are very weak

No protein is required to physically remove the RNA from the DNA

This type of termination is also called intrinsic

Stabilizes the RNA pol pausing

Many of the basic features of gene transcription are very similar in bacteria and eukaryotes

However, gene transcription in eukaryotes is more complex Larger organisms and cells Cellular complexity such as organelles

added complexity means more genes Multicellularity: many different cell types

increased regulation to express only in right cells at right time


12.3 TRANSCRIPTION IN EUKARYOTES

12-31


Nuclear DNA is transcribed by three different RNA polymerases RNA pol I

Transcribes all rRNA genes (except for the 5S rRNA) RNA pol II

Transcribes all structural genes Thus, synthesizes all mRNAs

Transcribes some snRNA genes RNA pol III

Transcribes all tRNA genes And the 5S rRNA gene

Eukaryotic RNA Polymerases

12-32


Eukaryotic promoter sequences are more variable and often much more complex than those of bacteria

For structural genes, at least three features are found in most promoters Regulatory elements TATA box (present in ~20 % of our genes) and other

short sequences in TATA-promoters that have a similar function

Transcriptional start site

Refer to Figure 12.13

Sequences of Eukaryotic Structural Genes

12-34

12-35

Usually an adenine

The core promoter is relatively short It consists of the TATA box

Important in determining the precise start point for transcription

The core promoter by itself produces a low level of transcription

This is termed basal transcription

Figure 12.13


12-36

Figure 12.13

Regulatory elements affect the binding of RNA polymerase to the promoter They are of two types

Enhancers Stimulate transcription

Silencers Inhibit transcription

They vary widely in their locations, from –50 to –100 region



Factors that control gene expression can be divided into two types, based on their “location”

cis-acting elements DNA sequences that exert their effect only over a

particular gene Example: TATA box, enhancers and silencers

trans-acting elements Regulatory proteins that bind to such DNA sequences

Sequences of Eukaryotic Structural Genes

12-37


Three categories of proteins are required for basal transcription to occur at the promoter RNA polymerase II Five different proteins called general transcription factors

(GTFs) A protein complex called mediator

RNA Polymerase II and its Transcription Factors

12-38

12-39

Figure 12.14


12-40

Figure 12.14


A closed complex

Released after the open complex is

formed

RNA pol II can now proceed to the

elongation stage


Basal transcription apparatus RNA pol II + the five GTFs

The third component for transcription is a large protein complex termed mediator It mediates interactions between RNA pol II and various

regulatory transcription factors

Its subunit composition is complex and variable

Mediator appears to regulate the ability of TFIIH to phosphorylate CTD

Therefore it plays a pivotal role in the switch between transcriptional initiation and elongation

12-41


The compaction of DNA to form chromatin can be an obstacle to the transcription process

Most transcription occurs in interphase Then, chromatin is found in 30 nm fibers that are

organized into radial loop domains Within the 30 nm fibers, the DNA is wound around histone

octamers to form nucleosomes

Chromatin Structure and Transcription

12-43


The histone octamer is roughly five times smaller than the complex of RNA pol II and the GTFs

The tight wrapping of DNA within the nucleosome inhibits the function of RNA pol

To circumvent this problem, the chromatin structure is significantly loosened during transcription

Two common mechanisms alter chromatin structure

Chromatin Structure and Transcription

12-44


1. Covalent modification of histones Amino terminals of histones are modified in various ways

Acetylation; phosphorylation; methylation

12-45

Figure 12.15

Adds acetyl groups, thereby loosening the interaction

between histones and DNA

Removes acetyl groups, thereby restoring a tighter interaction

These effects may significantly alter gene expression


2. ATP-dependent chromatin remodeling The energy of ATP is used to alter the structure of

nucleosomes and thus make the DNA more accessible

12-46

Figure 12.15

Proteins are members of the SWI/SNF family

Acronyms refer to the effects on yeast when these enzyme are

defectiveMutants in SWI are defective in

mating type switching

Mutants in SNF are sucrose non-fermenters

‘promoter’ Protein coding

Difference in gene structure between

- prokaryote

- eukaryotecore

‘promoter’

An important difference between prokaryotes and eukaryotes is that eukaryotes’ genes are not split into intons and exons in eukaryotes is the DNA coding protein are. Therefore, exons eventually end up in the mRNA

intron

exons

Pre-mRNA

Transcription start, elongation, termination and RNA processing in eukaryotes

: coding protein: non-coding protein: ‘leader’ and ‘trailer’

CAP

CAP (poly A tail)

The longest gene in human genome is more than 1.500.000 base pares (bp) and the mRNA is ~ 7000 nt. That means: >1.493.000 bp intron = ~ 99,5 % !!!!!

‘promoter’

intron

exons

GENE

mRNA AAAAAAAAAAAAAAn

Instead, coding sequences, called exons, are interrupted by intervening sequences or introns

Transcription produces the entire gene product Introns are later removed or excised Exons are connected together or spliced

This phenomenon is termed RNA splicing It is a common genetic phenomenon in eukaryotes Occurs occasionally in bacteria as well


12.4 RNA MODIFICATION

12-48

Aside from splicing, RNA transcripts can be modified in several ways For example

Trimming of rRNA and tRNA transcripts 5’ Capping and 3’ polyA tailing of mRNA transcripts


12.4 RNA MODIFICATION

12-49


Figure 12.20

In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA

Also as heterogeneous nuclear RNA (hnRNA)

This RNA is altered by splicing and other modifications, before it leaves the nucleus

Splicing in this case requires the aid of a multicomponent structure known as the spliceosome


The spliceosome is a large complex that splices pre-mRNA

It is composed of several subunits known as snRNPs (pronounced “snurps”) Each snRNP contains small nuclear RNA and a set of

proteins

Pre-mRNA Splicing

12-67


The subunits of a spliceosome carry out several functions

1. Bind to an intron sequence and precisely 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

Pre-mRNA Splicing

12-68


Figure 12.21

Intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries

The consensus sequences for the splicing of mammalian pre-mRNA are shown in Figure 12.21

Sequences shown in bold are highly conserved

Corresponds to the boxed adenine in Figure 12.22

Serve as recognition sites for the binding of the spliceosome

12-70

Intron loops out and exons brought closer

together

Figure 12.22Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display


Intron will be degraded and the snRNPs used again


One benefit of genes with introns is a phenomenon called alternative splicing

A pre-mRNA with multiple introns can be spliced in different ways This will generate mature mRNAs with different

combinations of exons

This variation in splicing can occur in different cell types or during different stages of development

Intron Advantage?

12-72


The biological advantage of alternative splicing is that two (or more) polypeptides can be derived from a single gene

This allows an organism to carry fewer genes in its genome

Intron Advantage?

12-73


Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end This event is known as capping

Capping occurs as the pre-mRNA is being synthesized by RNA pol II Usually when the transcript is only 20 to 25 bases long

Capping: marking 5’ends of mRNAs

12-74


The 7-methylguanosine cap structure is recognized by cap-binding proteins

Cap-binding proteins play roles in the

Movement of some RNAs into the cytoplasm Early stages of translation Splicing of introns

Function of Capping

12-77


Most mature mRNAs have a string of adenine nucleotides at their 3’ ends This is termed the polyA tail

The polyA tail is not encoded in the gene sequence It is added enzymatically after the gene is completely

transcribed

The 3’ end of a mRNA: Tailing

12-78


Figure 12.24

Consensus sequence in higher eukaryotes

Appears to be important in the transport and stability of mRNA

and the translation of the polypeptide

Length varies between species

From a few dozen adenines to several hundred

transcription and splicing

Documents

mcgrawhill companies

dna transcription

rate of rna transcription

rna transcripts

initiation of transcription

steps of transcription

transcription mrna sequences

rna polymeraseholoenzymeis